COMPOUNDS THAT TARGET TEM8, COMPOSITIONS, AND METHODS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application No. 63/306,165, filed February 3, 2022, which is incorporated herein by reference in its entirety.

GOVERNMENT FUNDING

This invention was made with government support under CA197292 and CAI 11412 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

This application contains a Sequence Listing electronically submitted to the United States Patent and Trademark Office via Patent Center as an XML file entitled

“0110.000689W001” having a size of 40 kilobytes and created on February 3, 2023. Due to the electronic filing of the Sequence Listing, the electronically submitted Sequence Listing serves as both the paper copy required by 37 CFR § 1.821(c) and the CRF required by § 1.821(e). The information contained in the Sequence Listing is incorporated by reference herein.

SUMMARY

This disclosure describes, in one aspect, a multispecific anti-TEM8 compound that generally includes an anti-TEM8 targeting domain and an immune cell engaging domain.

Generally, the anti-TEM8 targeting domain includes amino acids 277-390 of SEQ ID NO: 1, amino acids 277-389 of SEQ ID NO:2, amino acids 277-389 of SEQ ID NO:3, amino acids 277-389 of SEQ ID NO:4, amino acids 277-389 of SEQ ID NO:5, amino acids 277-515 of SEQ ID NO:25, or a TEM8-binding variant or fragment of any of the foregoing. In one or more embodiments, the TEM8-binding variant or fragment of SEQ ID NO: 1 comprises one, two, or three of SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28. In one or more embodiments, the TEM8-binding variant or fragment of SEQ ID NO:2 comprises one, two, or three of SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31. In one or more embodiments, the TEM8-binding variant or fragment of SEQ ID NO:3 comprises one, two, or three of SEQ ID NO:32, SEQ ID NO:27, or SEQ ID NO:33. In one or more embodiments, the TEM8-binding variant or fragment of SEQ ID NO:4 comprises one, two, or three of SEQ ID NO:34, SEQ ID NO:35, or SEQ ID NO:36. In one or more embodiments, the TEM8-binding variant or fragment of SEQ ID NO:5 comprises one, two, or three of SEQ ID NO:37, SEQ ID NO:38, or SEQ ID NO:39.

In one or more embodiments, the immune cell engaging domain engages a natural killer (NK) cell. In some of these embodiments, the immune cell engaging domain comprises a ligand or antibody that specifically binds to CD 16.

In one or more embodiments, the targeting domain and the immune cell engager domain are linked by any one of SEQ ID NOs:8-20.

In one or more embodiments, the multispecific anti-TEM8 compound further includes an immune cell activating domain. In some of these embodiments, the immune cell engaging domain engages an NK cell and the immune cell activating domain includes a cytokine or an NK activating portion or variant thereof.

In one or more embodiments, the immune cell engaging domain and the immune activating domain are linked by any one of SEQ ID NOs:8-20 and the immune cell activating domain and the targeting domain are linked by any one of SEQ ID NOs:8-20.

In one or more embodiments, the multispecific anti-TEM8 compound is as set forth as in any one of SEQ ID NO: 1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:25.

In another aspect, this disclosure describes an isolated nucleic acid sequence encoding any embodiment of the multispecific anti-TEM8 compound summarized above.

In another aspect, this disclosure describes a host cell that includes a nucleic acid sequence encoding a multispecific anti-TEM8 compound.

In another aspect, this disclosure describes a pharmaceutical composition that includes a multispecific anti-TEM8 compound and a pharmaceutically acceptable carrier.

In another aspect, this disclosure describes a method that includes administering to a subject a multispecific anti-TEM8 compound in an amount effective to induce natural killer (NK)-mediated killing of a cell.

In another aspect, this disclosure describes a method for stimulating expansion of natural killer (NK) cells in vivo. Generally, the method includes administering to a subject a multispecific anti-TEM8 compound in an amount effective to stimulate expansion of NK cells in the subject.

In another aspect, this disclosure describes a method for treating a subject having, or at risk of having cancer. Generally, the method includes administering to the subject a multispecific anti-TEM8 compound in an amount effective to ameliorate at least one symptom or clinical sign of cancer or decrease the likelihood that the subject develops cancer compared to an untreated individual.

In some of these embodiments, tumor cells or cells in tumor microenvironment of the cancer express TEM8.

In some of these embodiments, the cancer can be prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, or hematopoietic cancer.

In some of these embodiments, the multispecific anti-TEM8 compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy. In some of these embodiments, the chemotherapy includes altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, or vinorelbine.

The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. caml615TEM8 Tri-Specific Killer Engager (TriKE) construct and isolation. (A) caml615TEM8 includes a camelid anti-CD16 single domain antibody (sdAb), an IL- 15 moiety, and an anti-TEM8 scFv. (B) A representative GelCode Blue-stained polyacrylamide gel of caml615TEM8 isolated from Expi293F supernatant and the corresponding Image! densitometry plot used to calculate purity. The desired product is indicated with an arrow at approximately 60 kDa (predicted molecular weight = 55.7 kDa) and the major peak. Impurities are identified with arrows at the faint bands and minor peaks. The calculated purity of the representative product was 88.76%.

FIG. 2. caml615TEM8 Tri-Specific Killer Engager (TriKE) induction ofNK cell degranulation and cytokine production against TEM8 ⁺ tumor cell lines. caml615TEM8- mediated NK cell degranulation and IFNy production dose-response curves, and representative CD107a vs IFNy FlowJo plots of the highest concentration tested — 9 nM caml615TEM8. caml615TEM8 was serially diluted 1 :3 from 9 nM down to 1.37 pM. (A) Without target cells (n=4, fresh PBMCs). (B) Against TEM8 ⁺ targets, A549 (n=7, fresh PBMCs) and DU145 (n=5, frozen PBMCs), and against TEM8" targets, HT-29 (n=5, frozen PBMCs) and Raji (n=4, fresh PBMCs).

FIG. 3. caml615TEM8 Tri-Specific Killer Engager (TriKE) induction ofNK cell degranulation and cytokine production against TEM8 ⁺ tumor cell lines. (A) Effect of TEM8 blocking with anti-TEM8 scFv on NK cell degranulation for TEM8 ⁺ and TEM8" tumor cell lines at 9 nM caml615TEM8 and targets blocked with 1800 nM anti-TEM8 scFv (n=4, frozen PBMCs for all tumor cell lines in TEM8 blocking experiments). (B) Effect of TEM8 blocking with anti-TEM8 scFv on IFNy production for TEM8 ⁺ and TEM8" tumor cell lines at 9 nM caml615TEM8 and targets blocked with 1800 nM anti-TEM8 scFv (n=4, frozen PBMCs for all tumor cell lines in TEM8 blocking experiments).

FIG. 4. caml615TEM8 induction ofNK cell degranulation and cytokine production against TEM8 ⁺ endothelial and fibroblast lines. caml615TEM8-mediated NK cell degranulation and cytokine production dose-response curves, and representative CD 107a versus IFNy FlowJo plots of the highest concentration tested — 9 nM caml615TEM8. caml615TEM8 was serially diluted 1 :3 from 9 nM down to 1.37 pM. (A) Against TEM8 ⁺ endothelial cell lines: Human Umbilical Vein Endothelial Cells (HUVECs, n=7, frozen PBMCs), Human Microvascular Endothelial Cells from Lung (HMVEC-Ls, n=4, frozen PBMCs), and the immortalized mouse endothelial cell line 2H-11 (n=4, fresh PBMCs). (B) Against TEM8 ⁺ fibroblasts: Normal Human Lung Fibroblasts (NHLFs, n=5, fresh PBMCs).

FIG. 5. caml615TEM8 induction of NK cell degranulation and cytokine production against TEM8 ⁺ endothelial and fibroblast lines. (A) Effect of TEM8 blocking with anti-TEM8 scFv on NK cell degranulation for TEM8 ⁺ endothelial and fibroblast lines at 9 nM caml615TEM8 and targets blocked with 1800 nM anti-TEM8 scFv (n=4, frozen PBMCs for all stromal cell lines in TEM8 blocking experiments). (B) Effect of TEM8 blocking with anti-TEM8 scFv on IFNy production for TEM8 ⁺ endothelial and fibroblast lines at 9 nM caml615TEM8 and targets blocked with 1800 nM anti-TEM8 scFv (n=4, frozen PBMCs for all stromal cell lines in TEM8 blocking experiments).

FIG. 6. caml615TEM8 stimulates superior NK cell-mediated killing of TEM8 ⁺ tumor spheroids compared to IL-15. (A) Real-time analysis of enriched NK cells from a representative donor killing A549 NucLight Red labeled spheroids alone or in the presence of 3 nM caml615TEM8 or IL-15. Data points are displayed at every four hours. (B) Overlaid brightfield and red fluorescence images of A549 spheroids co-incubated with the representative donor at the specified timepoints for each treatment. (C) Pooled data of A549 tumor spheroid areas on Day 0, Day 2, Day 4, and Day 6. The horizontal bar at each timepoint represents the average spheroid size of A549 only wells.

FIG. 7. caml615TEM8 does not stimulate superior NK cell-mediated killing of TEM8" tumor spheroids compared to IL-15. (A) Real-time analysis of enriched NK cells from a representative donor killing HT-29 NucLight Red labeled spheroids alone or in the presence of 3 nM caml615TEM8 or IL-15. Data points are displayed at every four hours. (B) Overlaid brightfield and red fluorescence images of HT-29 spheroids co-incubated with the representative donor at the specified timepoints for each treatment. (C) Pooled data of HT-29 tumor spheroid areas on Day 0, Day 2, Day 4, and Day 6. The horizontal bar at each timepoint represents the average spheroid size of A549 only wells.

FIG. 8. caml615TEM8 selectively stimulates proliferation ofNK cells rather than T cells. (A) Representative FlowJo proliferation graphs of CellTrace Violet stained NK cells and T cells in response to seven-day incubation without treatment, in 9 nM IL- 15, or in 9 nM cam!615TEM8. FIG. 9. caml615TEM8 selectively stimulates proliferation ofNK cells rather than T cells. IL-15-mediated and caml615TEM8-mediated NK cell proliferation and T cell proliferation dose response curves. Treatments were serially diluted 1 :3 from 9 nM down to 1.37 pM. (A) “Proliferated” cells were defined as having proliferated at least once. (B) “Highly Proliferated” cells were defined as having proliferated at least twice.

FIG. 10. caml615TEM8 selectively stimulates pSTAT5 induction in NK cells rather than T cells. pSTAT5 induction in NK and T cells following a 20-minute incubation in IL-15 or caml615TEM8. Treatments were serially diluted 1 :3 from 9 nM down to 1.37 pM.

FIG. 11. caml615TEM8 slows tumor growth compared to functionally equivalent IL-15. (A) Timeline of the in vivo assay with indicated timepoints. (B) Left: Tumor growth curves depicting tumor volume from Day 7 to Day 42 for individual male mice (filled in triangles and diamonds) and female mice (empty triangles and diamonds). Right: Combined tumor volume data depicting the overall mean tumor volume for all mice treated with either functionally equivalent IL-15 or caml615TEM8. Tumor volumes were compared at Day 42.

FIG. 12. caml615TEM8 improves survival and enhances NK cell expansion compared to functionally equivalent IL-15. (A) Kaplan Meier Survival curves depicting the percent surviving for mice receiving each treatment. (B) NK cell count in 100 pl of mouse peripheral blood on Day 28. Data for Donor 1 and Donor 2 (in male and female mice, respectively) are displayed and analyzed separately.

FIG. 13. caml615TEM8 increases NK tumor infiltration compared to functionally equivalent IL-15. (A) Top: Representative Granzyme B stained tumor on Day 23 at 240X magnification. Bottom: The Granzyme B mask used for NK identification generated with cell detection software (QuPath; Bankhead et al., Scientific Reports 7: 16878, 2017). (B) Left: Dot plots of Granzyme B ⁺ cell counts from seven frames in individual tumors. The black bar is the mean for that mouse. Right: Combined data showing the overall mean number of Granzyme B ⁺ cells per frame for tumors from all mice.

FIG. 14. caml615TEM8 reduces endothelial density compared to functionally equivalent IL-15. (A) Top: Representative % CD31 ⁺ stained tumor on Day 42 at 120X magnification to evaluate endothelial density for functionally equivalent IL-15-treated versus caml615TEM8- treated tumors. Bottom: The CD31 mask used for NK identification generated with cell detection software (QuPath; Bankhead et al., Scientific Reports 7: 16878, 2017). (B) Left: Dot plots of percentage of CD31 ⁺ area from seven frames in individual tumors. The black bar is the mean for that mouse. Right: Combined data showing the overall mean percentage of CD31 ⁺ area for tumors from all mice.

FIG. 15. caml615TEM8-treated NK cells mediate improved tumor control compared to caml615TEM8 without NKs. (A) Tumor growth curves depicting tumor volume data from Day 0 to Day 42 for individual male mice. (B) Combined tumor volume data depicting the overall mean tumor volume for all male mice treated. Tumor volumes were compared at Day 42. (C) Kaplan Meier Survival curve depicting the percent surviving for mice receiving each treatment. One mouse is censored. Below are the statistical analyses of median survival.

FIG. 16. caml615TEM8-treated NK cells mediate improved tumor control compared to molecularly equivalent IL-15-stimulated NK cells. (A) Tumor growth curves depicting tumor volume data from Day 0 to Day 42 for individual female mice. (B) Combined tumor volume data depicting the overall mean tumor volume for all female mice treated. Tumor volumes were compared at Day 42. (C) The number of NK cells in 100 pl of mouse peripheral blood on Day 28 for female mice treated with Donor 2.

FIG. 17. Knocking out ANTXR1 on A549s. (A) A CRISPR/Cas9 system was designed to knock out exon 1 of the ANTXR1 gene. Forward and reverse PCR primers were designed to anneal upstream and downstream of the predicted cleavage sites. (B) Knockout status of single cell clones was determined by PCR, where amplification of the wild-type ANTXR1 gene creates a product of approximately 1,700 base pairs while amplification of the desired exon 1 knockout mutant allele creates a product of approximately 900 base pairs. Representative images of the wild type homozygote (TEM8+/+), wild type/knockout heterozygote (TEM8+/-), and knockout homozygote (TEM8-/-) PCR products.

FIG. 18. Knocking out ANTXR1 on A549s abolishes caml615TEM8-mediated NK cell activity against A549s. caml615TEM8-mediated NK cell degranulation and IFNy production dose-response curves against A549 TEM8-/-, and representative CD 107a vs IFNy plots of the highest concentration tested — 9 nM caml615TEM8. caml615TEM8 was serially diluted 1 :3 from 9 nM down to 1.37 pM. (n=4).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS This disclosure describes compositions and methods that include a TEM8-targeting compound. As sued herein, “TEM8-targeting,” “anti-TEM8,” and “TEM8-binding” are used interchangeably to refer to compounds that specifically bind to TEM8. Generally, the TEM8- targetingcompounds described herein include a TEM8-targeting domain. In one or more embodiments, the TEM8-targeting compound can include one ore more additional functional domains. When present, the additional functional domain or domains may be directly or indirectly operably linked to the anti-TEM8 domain by flanking sequences.

Cancer immunotherapies for solid tumors need to overcome the cancer-promoting stromal cells within the tumor microenvironment, including cancer-associated fibroblasts, endothelial cells, pericytes, and immune cells. Tumor stromal cells abet cancer growth via growth factors and cause immunosuppression and immune cell dysfunction. Finally, the tumor vasculature, although abnormal, disorganized, and dilated, supplies cancer cells with the nutrients and oxygen necessary for tumor growth and dissemination.

Current antiangiogenic therapies are plagued by limited efficacy and toxicities. Efficacy is limited because angiogenic signaling redundancies result in tumor resistance. Toxicity occurs because the targeted pathways are important in normal adult angiogenesis. Preclinical attempts to target other stromal cell types, like cancer-associated fibroblasts, have similarly encountered on target, off tumor toxicities due to target protein expression in healthy tissue.

Tumor Endothelial Marker 8 (TEM8, encoded by the ANTXR1 gene) is a highly conserved 80-85 kDa integrin-like adhesion molecule that was discovered on the endothelium of colorectal cancer, and subsequent studies identified its expression on multiple stromal cells — endothelial cells, fibroblasts, and pericytes — in the tumor microenvironment (TME) of diverse human cancer types (Chaudhary et al., Cancer Cell 21(2):212-226; Szot et al., J Clin Invest 128(7):2927-2943, 2018). TEM8 expression also has been described on diverse cancer cell lines in vitro and has been implicated on breast cancer and pancreatic cancer stem cells. TEM8 is unique as it is not expressed on healthy tissue or during normal angiogenesis, indicating that TEM8 expression is unique to the tumor microenvironment (Szot et al., J Clin Invest 128(7):2927-2943, 2018) Preclinical approaches targeting TEM8 with TEM8 blocking antibodies, TEM8-MMAE antibody-drug conjugates, and TEM8 CAR T cells have demonstrated low toxicities (Chaudhary et al., Cancer Cell 21(2):212-226; Szot et al., J Clin Invest 128(7):2927-2943, 2018; Byrd et al., Cancer Res 78(2):489-500). Natural Killer (NK) cells are a subset of lymphocytes with cytolytic activity against virally infected and malignantly transformed cells, and thus are involved in host defenses against cancer. NK cells express activating receptors and inhibitory receptors whose signals are integrated to determine NK cell killing. NK cell activity is also regulated by cytokines, particularly IL-2 and IL- 15, that stimulate NK cell proliferation and activation. Numerous therapies employ NK cells to combat cancer, including monoclonal antibodies, allogeneic NK cells, and cytokine therapy with recombinant IL-15 and IL-15 superagonist complexes.

Multispecific Killer engager compounds have functional domains that form a cytolytic bridge between NK cell and target: an immune cell engaging domain, an NK activating domain that induces NK proliferation and activation, and an antigen-specific targeting domain (Vallera et al., Clin Cancer Res 22(14):3440-3450; US Patent No. 11,098,100; US Patent No. 11,098,101; US Patent Application Publication No. US 2022/0324972 Al; International Patent Application Publication No. WO 2021/076545 Al; International Patent Application Publication No. WO 2021/247794 A2; and International Patent Application Publication No. WO 2022/150379 Al). This disclosure describes novel multispecific Killer engager compounds that target NK cells against TEM8-expressing tumor and tumor stroma.

The design of an exemplary TEM8-targeting tri-specific Killer engager compound is shown schematically in FIG. 1 A. The illustrated exemplary TEM8-targeting tri-specific Killer engager compound (caml615TEM8) includes a camelid single domain antibody that binds to NK cells and stimulates CD16 on NK cells, an IL-15 moiety that stimulates IL-15 signaling on NK cells, and a single chain variable fragment (scFv) that specifically binds to TEM8 to mediate antigen-specific target cell recognition. caml615TEM8 was isolated from Expi293F supernatant by a C-terminal 10X histidine tag, resulting in a highly pure protein product of about 60 kDa (predicted molecular weight = 55.7 kDa, FIG. IB).

While described herein in the context of an exemplary embodiment in which a TEM8- targeting multispecific Killer engager compound is the tri-specific Killer engager caml615TEM8, the compositions and methods described herein can involve a TEM8-targeting compound lacking an immune cell engaging domain or having an alternative and/or additional immune cell engaging domain, lacking an immune cell activating domain or having an alternative and/or additional immune cell activating domain, an alternative and/or additional TEM8-binding domain, and/or an alternative flanking amino acid sequence, as described in more detail below. caml615TEM8 induces dose-dependent NK cell degranulation and IFNy production against TEM8+ tumor targets.

PBMCs were incubated with tumor target cells at a range of caml615TEM8 concentrations to evaluate caml615TEM8-mediated NK cell degranulation (CD 107a) and inflammatory cytokine (IFNy) production. The A549 (Non-small cell lung) and DU145 (prostate) cell lines highly express ANTXR1 and were designated TEM8 ⁺ cell lines; the HT-29 (colon) and Raji (Burkitt’s lymphoma) cell lines do not express ANTXR1 and were designated TEM8" cell lines. caml615TEM8 induced no NK cell degranulation or IFNy production in the absence of target cells (FIG. 2A). caml615TEM8 induced strong NK cell degranulation and IFNy production against A549 and DU145 TEM8 ⁺ tumor cell lines, but almost no NK cell degranulation or cytokine production against HT-29 or Raji TEM8" tumor cell lines (FIG. 2B), indicating that target cell TEM8 expression is necessary for caml615TEM8-mediated NK cell degranulation and cytokine production.

TEM8 blockade, through pre-incubation with excess anti-TEM8 scFv, resulted in significantly less caml615TEM8-induced NK cell degranulation and IFNy production against TEM8 ⁺ lines, whereas no significant reduction in NK degranulation or IFNy production was seen against TEM8" lines (FIG. 3). Finally, an A549 TEM8 knockout cell line was created using A CRISPR/Cas9 system designed to knock out exon 1 of ANTXR1 (FIG. 17A). The knockout status of single cell clones was tested by polymerase chain reaction, and a homozygous knockout clone was identified — hereafter, A549 TEM8' ^/_ (FIG. 17B). Knocking out TEM8 eliminated caml615TEM8-mediated NK cell degranulation and cytokine production against A549s (FIG. 18), further showing specificity. caml615TEM8 induces NK cell degranulation and IFNy production against endothelial cells and fibroblasts

TEM8 has been identified on multiple stromal cells within the tumor microenvironment of diverse human cancer types (Chaudhary et al., Cancer Cell 21(2):212-226; Szot et al., J Clin Invest 128(7):2927-2943, 2018). TEM8 is upregulated on some stromal cell lines in vitro, thereby providing an opportunity to study NK -mediated toxicity against stromal cells that have been shown to express the TEM8 only within the tumor microenvironment. While NK cell cytotoxicity towards tumor cells is well described, the ability to target NK cells against non- cancerous stromal cells is less clear. For example, the Human Umbilical Vein Endothelial Cell (HUVEC) line expresses low levels of TEM8, and caml615TEM8 stimulated modest NK cell degranulation and IFNy production against HUVECs (FIG. 4A). caml615TEM8 induced more robust NK cell degranulation and IFNy production against Human Microvascular Endothelial Cells from Lung— HMVEC-L (FIG. 4A).

Mouse TEM8 is highly homologous to human TEM8, and the TEM8-engaging scFv sequence within caml615TEM8 binds to both human and mouse. The 2H-11 cell line is a relevant murine model for tumor endothelial cells because they display activity in standard endothelial function assays and express murine homologs for standard human endothelial markers, including TEM8. caml615TEM8 stimulated strong NK cell degranulation and IFNy production against 2H-1 Is (FIG. 4 A), indicating that activity against murine tumor stroma could be measured in a xenogeneic model.

TEM8 is also expressed by cancer-associated fibroblasts, which contribute to tumor growth and affect immune activity (Vitale et al., Eur J Immunol 44: 1582-1592, 2014; Szot et al., J Clin Invest 128(7):2927-2943, 2018). caml615TEM8 potently induced NK cell degranulation and IFNy production against Normal Human Lung Fibroblasts — NHLFs (FIG. 4B), indicating that caml615TEM8 can target NK cells against multiple TEM8 ⁺ stromal cell types present within the tumor microenvironment. Blocking TEM8 resulted in significantly less caml615TEM8-induced NK cell degranulation and IFNy production against these cell lines (FIG. 5). caml615TEM8 stimulates NK cell killing of TEM8 ⁺ tumor spheroids

Since caml615TEM8 preferentially induces NK cell degranulation and cytokine production against TEM8 ⁺ tumor cell lines, evaluation if this would translate to killing within the more stringent and physiologic tumor spheroid model was undertaken. NucLight Red-expressing A549 (TEM8 ⁺ ) spheroids were formed and treated with enriched NK cells alone or in the presence of caml615TEM8 or IL-15. caml615TEM8 resulted in more rapid and complete A549 spheroid elimination than equimolar IL- 15 (FIG. 6). In contrast, cam!615TEM8 did not improve NK cell-mediated killing of NucLight Red-expressing HT-29 (TEM8‘) spheroids compared to IL- 15 (FIG. 7).

The camelid anti-CD16 nanobody, within caml615TEM8 TriKE, enhances IL- 15 signaling specifically in NK cells

IL- 15 stimulates NK cell activation and survival by signaling through the IL- 15 receptor complex on NK cells. The IL- 15 signaling capacity of caml615TEM8 was evaluated in a proliferation assay. Briefly, whole PBMCs were stained with CELLTRACE VIOLET (Thermo Fisher Scientific, Inc., Waltham, MA) and incubated with IL-15 or caml615TEM8 for seven days, at which time proliferation of live NK and T cells was assessed by flow cytometry (FIG. 8). NK cell proliferation was similar between IL- 15 and caml615TEM8 at all concentrations, with both treatments inducing strong, dose-dependent NK proliferation (FIG. 9A). However, T cells proliferated substantially less when compared to IL-15 (FIG. 9A). The caml615TEM8 induced more “highly proliferated” NK cells — defined as two or more cell divisions — than IL- 15, while IL-15 induced more highly proliferated T cells than caml615TEM8 (FIG. 9B). To further understand the differential in signaling from caml615TEM8 on NK cells versus T cells, STAT5 phosphorylation, mediated by IL-15 or caml615TEM8 stimulation, was evaluated. IL-15 and caml615TEM8 induced similar levels of pSTAT5 in NK cells, whereas IL- 15 stimulated higher levels of pSTAT5 than caml615TEM8 in T cells (FIG. 10). caml615TEM8 stimulates in vivo NK cell control of A549 tumors

Based on the TEM8-specific NK cell targeting of tumor and stroma in vitro, the ability of caml615TEM8 to stimulate NK cell targeting of TEM8-expressing tumors and tumor endothelium in vivo was evaluated next. A mouse model was used for these studies. Murine TEM8 is highly homologous to human TEM8, expression of murine TEM8 is absent or very low in normal mouse tissues, and murine TEM8 is upregulated on the vasculature of human tumor xenografts in mice. Moreover, FIG. 4A (right) shows caml615TEM8 activates NK against murine endothelial cells, indicating that targeting of endothelium can be modeled in the xenograft system despite absence of human stroma.

A549/GFP/Luc tumors (TEM8 ⁺ ) were engrafted in NSG mice, followed by injection of expanded NK cells two weeks later, and treatment with caml615TEM8 or functionally equivalent IL-15. Mice were assessed for a number of parameters during the course of several studies with this setup (FIG. 11 A). On Day 42, tumors from male and female mice treated with NKs and caml615TEM8 were significantly smaller than tumors treated with NKs and functionally equivalent IL- 15 (FIG. 1 IB). Mice treated with NKs and caml615TEM8 also survived longer than mice treated with NKs and functionally equivalent IL- 15 (Median survival, NK+EF IL-15: 49 days versus NK+caml615TEM8: 64 days) (FIG. 12A). caml615TEM8 was also tested in the absence of NK cells to control for caml615TEM8 NK-independent effects, as TEM8 masking antibodies have been shown to reduce human tumor xenograft growth in previous studies, but caml615TEM8 alone did not induce an effect (FIG. 15). Equimolar IL- 15 was also tested to determine whether NKs with higher doses of IL- 15 demonstrated improved tumor control, but not significant differences were seen with functionally equivalent IL- 15 and the caml615TEM8 treatment was still significantly better (FIG. 16). When circulating NK cell numbers were evaluated within the mice at Day 28, mice treated with caml615TEM8 had reproducibly greater NK cell numbers than mice treated with functionally equivalent IL-15 in two separate experiments (FIG. 12B). caml615TEM8 also mediated increased NK cell numbers compared to equimolar IL- 15, while the two IL- 15 doses did not differ from each other (FIG. 16C). In summary, caml615TEM8 stimulated superior NK-mediated tumor control than IL- 15 in vivo. caml615TEM8 stimulates better tumor infiltration and reduces endothelial density on A549 tumors

To determine whether caml615TEM8 would also stimulate enhanced NK cell tumor infiltration in the A549 xenograft model, Day 23 tumors were harvested and analyzed by immunohistochemistry (IHC) for anti-human Granzyme B to detect human NK cell (the only cells containing this human marker withing this model). A positive cell mask was developed using cell detection software (QuPath; Bankhead et al., Scientific Reports 7: 16878, 2017) to quantify tumor-infiltrating human NK cells in response to caml615TEM8 or functionally equivalent IL-15 (FIG. 13 A). caml615TEM8 treatment resulted in increased NK cell tumor infiltration compared to functionally equivalent IL- 15 (FIG. 13B).

To determine whether caml615TEM8 would stimulate an NK cell-mediated reduction in tumor endothelial density, indicating that NKs were being directed to the mouse-derived TEM8 expressed on tumor endothelium, Day 42 tumors were harvested and analyzed by immunohistochemistry for anti-mouse CD31. QuPath cell detection software was used to quantify the percent CD31 ⁺ area in digital images of the areas with the highest endothelial density (FIG. 14A). Tumors treated with NKs and caml615TEM8 had significantly lower endothelial density compared to tumors treated with NKs and functionally equivalent IL- 15 (FIG. 14B). This data indicates that caml615TEM8 decreased endothelial density by about three-fold, suggesting that TEM8 targeted NK cells can kill both cancer and the cancerized stroma, a dual mechanism felt to be important in solid tumors.

Thus, this disclosure describes multispecific NK engager compounds that target NK cells to TEM8, a cancer target expressed on tumor and tumor stromal cells. The exemplary multispecific NK engager compound, the tri-specific compound caml615TEM8, specifically stimulated NK cell degranulation and IFNy production better than equimolar IL- 15 against TEM8-expressing A549 and DU145 cell lines and induced rapid killing of TEM8 ⁺ A549 tumor spheroids, which mimic features of solid tumors. The tri-specific compound caml615TEM8 also induced better A549 tumor control in a xenogeneic mouse model. Therefore, CD16 agonism with TEM8 engagement by caml615TEM8 stimulates NK cell killing beyond the cytokine- mediated NK cell activation by the IL- 15 moiety.

Previously described tri-specific Killer engager compounds only targeted NK cells to tumor cells, but since TEM8 is expressed on tumor endothelial cells and other tumor stromal cells, multispecific TEM8-targeted compounds also deliver therapeutics to these tissues. NK cell cancer immunotherapies for solid tumors need to overcome the cancer-promoting stromal cells within the tumor microenvironment, including cancer-associated fibroblasts, endothelial cells, pericytes, and immune cells, which can account for a significant portion of the tumor mass in many common carcinomas and contribute to NK cell dysfunction within the tumor through immunosuppression. Tumor stromal cells, e.g., fibroblasts, are implicated in intra-tumoral NK cell dysfunction, in, for example, in melanoma and breast cancer. Therefore, targeting tumorpromoting stromal cells may promote NK cell function rather than dysfunction within the tumor microenvironment. caml615TEM8 induced NK cell degranulation and IFNy production against TEM8 ⁺ endothelial cells and fibroblasts, showing NK cell immunotherapy can target tumor stroma. Despite the limited clinical efficacy and toxicides of currently approved anti angiogenic agents, this approach remains of interest for a variety of reasons. First, angiogenesis is necessary for solid tumor growth. Second, fibroblasts and pericytes are present in many solid tumors so anti-stromal therapies may be efficacious for diverse cancer histotypes. Third, eliminating the tumor endothelium may have a significant “domino effect” that kills many more cancer and tumor stromal cells deprived of oxygen and nutrients. Finally, tumor stromal cells are more genomically stable and less likely to undergo antigen loss and drug resistance than cancer cells. caml615TEM8 targeted NK cells to both TEM8 ⁺ tumor and tumor endothelium in an A549 xenograft model, resulting in smaller tumors and decreased tumor endothelium than treatment with IL-15. The striking decrease in endothelial density with caml615TEM8 treatment was likely a driving factor to the reduction in tumor growth.

Tumors in mice treated with caml615TEM8 had significantly more tumor-infiltrating NK cells, as assessed by the presence of human-Granzyme B expressing cells, than tumors in mice treated with functionally equivalent IL-15. Immune infiltration is generally considered a positive prognostic marker for immunotherapy outcomes. Greater NK cell tumor infiltration correlates with a better prognosis in diverse cancer types, but the effect of tumor-infiltrating NKs likely depends on NK expression of activating receptors, cancer cell expression of NK activating ligands, and/or the presence or absence of local immunomodulatory cells and molecules. Without wishing to be bound by any particular theory, multispecific therapeutic compounds that specifically bind TEM8 such as the exemplary tri-specific compound caml615TEM8 may increase tumor immunoreactivity by increasing the number of tumor-infiltrating NK cells, activating the infiltrating NK cells through the anti-CD16 nanobody and IL- 15 moiety, and/or stimulating NK cell killing of immunosuppressive stromal cell types within the microenvironment.

Novel cancer immunotherapies must consider the off-tumor and systemic effects of immune activation and the resultant toxicities. Trials evaluating the systemic administration of unmodified cytokines like IL-2, IL-15, or IFNy, although modestly effective in a subset of patients, were limited by toxicities. Accordingly, more selective delivery of immune activating cytokines may permit larger, more effective doses that result in greater anti-tumor activity and less systemic inflammation. The CD 16 sdAb immune cell engaging domain within cam!615TEM8 specifically enhanced signaling of the IL-15 moiety in CD16-expressing NK cells, resulting in a dose-dependent stimulation of NK cell proliferation without T cell proliferation. The exact mechanism of this preferential NK cell proliferation is unclear, but the preferential IL- 15 stimulation of NK cells and relative absence of IL- 15 stimulation of T cells may be an effective method of reducing dose-limiting toxicities. Furthermore, since TEM8 expression has been shown to be specific to tumor endothelium, therapies targeting TEM8 (e.g., the exemplary tri-specific caml615TEM8 compound) may have fewer on target, off tumor toxicities in humans than anti angiogenic therapies targeting traditional angiogenic pathways like VEGF. No caml615TEM8-related toxicities were observed in the in vivo studies described herein, despite cross-reactivity with mouse TEM8, consistent with other studies of TEM8- targeting preclinical therapies.

In summary, this disclosure describes multispecific therapeutic compounds that target TEM8 and demonstrates that an exemplary tri-specific TEM8-argeting compound, caml615TEM8, mediates not only NK-targeting of the tumor, but also mediates NK-targeting to the tumor support system and architecture. The target selectivity is coupled with selective delivery of IL-15 to NK cells, reducing toxicities. These data suggest that TEM8-targeting therapeutic compounds such as caml615TEM8 may be a valuable and novel anti -tumor, antistroma, and anti-angiogenic cancer therapy for patients with solid tumors.

In one or more embodiments, the TEM8-binding compound can be a multispecific compound. As used herein, the term “multispecific” refers to a compound (e.g., a polypeptide) possessing two or more binding domains, with or without a further effector molecule, covalently linked (e.g., fused) by recombinant, chemical, or other suitable method. A multispecific compound can include, as any domain with a binding function, an antibody, a binding fragment thereof, or binding variant thereof.

The term “antibody” refers to a molecule that contains at least one antigen binding site that immunospecifically binds to a particular antigen target of interest. The term “antibody” thus includes but is not limited to a full length antibody and/or its variants, a fragment thereof, a peptibody and variants thereof, a monoclonal antibody (including a full-length monoclonal antibody), a multispecific antibody (e.g., a bispecific antibody) formed from at least two intact antibodies, a human antibody, a humanized antibody, or an antibody mimetic that mimics the structure and/or function of an antibody or a specified fragment or portion thereof (e.g., a single chain antibody or fragment thereof). Thus, as used herein, the term “antibody” encompasses antibody fragments capable of binding to a biological molecule (such as an antigen or receptor) or a portion thereof, including but not limited to a Fab, a Fab' a F(ab')2, a pFc', a Fd, a single domain antibody (sdAb), a variable fragment (Fv), a single-chain variable fragment (scFv) or a disulfide-linked Fv (sdFv); a diabody or a bivalent diabody; a linear antibody; a single-chain antibody molecule; and a multispecific antibody (e.g., a tribody) formed from antibody fragments. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), or subclass.

Thus, in one aspect, this disclosure describes compounds that, in general terms, include a targeting domain that specifically binds to TEM8 and an immune cell engaging domain. Multispecific immune cell engagers may, for example, be used clinically to bridge two cells together (e.g., an effector cell such as a Natural Killer (NK) cell and a target cell such as a tumor cell) and induce activation of the effector cells to kill the target cell.

The targeting domain can include any moiety that selectively binds to a TEM8 ⁺ target such as, for example, a tumor cell, a target in the cancer stroma, or an immobilized TEM8 ⁺ cell. In one or more embodiments, the targeting domain can include an amino acid sequence. Thus, a targeting domain can include, for example, an anti-TEM8 antibody. In one or more embodiments, an anti-TEM8 antibody can include an anti-TEM8 polypeptide as described in detail herein. In one exemplary embodiment, the anti-TEM8 polypeptide can include one or more of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or a binding fragment or variant of any of the foregoing such as, for example, one or more complementarity-determining regions (CDRs). In one or more embodiments, the anti-TEM8 polypeptide can include a TEM8-binding fragment or variant of any one of SEQ ID NOs: 1-5. Suitable TEM8-binding fragments or variants can include one, two, or all three of the CDRs of any of SEQ ID NOs: 1-5. Thus, for example, the anti-TEM8 polypeptide can include a TEM8- binding fragment or variant of SEQ ID NO: 1 that includes one, two, or all three CDRs of SEQ ID NO: 1 (SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28); a TEM8-binding fragment or variant of SEQ ID NO:2 that includes one, two, or all three CDRs of SEQ ID NO:2 (SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31); a TEM8-binding fragment or variant of SEQ ID NO:3 that includes one, two, or all three CDRs of SEQ ID NO:3 (SEQ ID NO:32, SEQ ID NO:27, SEQ ID NO:33); a TEM8-binding fragment or variant of SEQ ID NO:4 that includes one, two, or all three CDRs of SEQ ID NO:4 (SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36); or a TEM8-binding fragment or variant of SEQ ID NO: 5 that includes one, two, or all three CDRs of SEQ ID NO:5 (SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39).

In one or more embodiments, the TEM8-binding compound can include one or more additional targeting domains beyond the TEM8-specific targeting domain. Additional targeting domains can increase binding specificity of the compound when, for example, a subset of TEM8 ⁺ cells is the desired target and/or when the use of the TEM8-binding compound includes bridging an immune cell with more than one target. Additional exemplary amino acid sequences suitable for use in the targeting domain are described in US Patent No. 11,098,100; US Patent No. 11,098,101; US Patent Application Publication No. 2020/0087369; US Patent Application Publication No. 2021/0395326; US Patent Application Publication No. US 2022/0324972 Al; International Patent Application Publication No. WO 2021/076545 Al; International Patent Application Publication No. WO 2021/247794 A2; and International Patent Application Publication No. WO 2022/150379 Al.

The immune cell engaging domain can include any moiety that binds to and/or activates an immune cell (e.g., an NK cell) and/or any moiety that blocks inhibition of an immune cell (e.g., an NK cell). In one or more embodiments, the immune cell engaging domain can include an amino acid sequence. In one or more embodiments, the immune cell engaging domain can include an antibody that selectively binds to a component of the surface of the immune cell. In other embodiments, the immune cell engaging domain can include a ligand or small molecule that selectively binds to a component of the surface of the immune cell. Thus, for brevity, reference to an antibody that selectively binds to a component of the surface of an immune cell includes any antibody fragment that exhibits the described binding character. Similarly, reference to a ligand that selectively binds to a component of the surface of an immune cell includes any fragment of the ligand that exhibits the described binding character.

While described herein in the context of exemplary embodiments in which the immune cell engaging domain engages an NK cell, the compositions and methods described herein can be modified to include an immune cell engaging domain that engages any desired immune cell or population of immune cells. For example, the multispecific anti-TEM8 compound can be designed to engage monocytes, macrophages, or T cells by selecting an immune cell engaging domain that engages the desired immune cell population. In one or more embodiments, the immune cell engaging domain can selectively bind to a receptor at least partially located at the surface of an immune cell. In certain embodiments, the immune cell engaging domain can serve a function of binding an immune cell and thereby bring the immune cell into spatial proximity with a target to which the targeting domain selectively binds. In certain embodiments, however, the immune cell engaging domain can selectively bind to a receptor that activates the immune cell and therefore also possess an activating function. As described above, activation of the CD 16 receptor can elicit antibody-dependent cell-mediated cytotoxicity in NK cells. Thus, in certain embodiments, an NK engaging domain can include at least a portion of an anti-CD16 receptor antibody effective to selectively bind to the CD 16 receptor. In other embodiments, an NK engaging domain may interrupt mechanisms that inhibit NK cells. In such embodiments, the NK engager domain can include, for example, anti- PD1/PDL1, anti-NKG2A, anti-TIGIT, anti-killer-immunoglobulin receptor (KIR), and/or any other inhibition blocking domain.

One can design an NK engaging domain to possess a desired degree of NK selectivity and, therefore, a desired immune engaging character. For example, CD 16 has been identified as Fc receptors FcyRIIIa (CD 16a) and FcyRIIIb (CD 16b). These receptors bind to the Fc portion of IgG antibodies that then activates the NK cell for antibody-dependent cell-mediated cytotoxicity. Anti-CD16 antibodies selectively bind to NK cells, but also can bind to neutrophils. Anti-CD16a antibodies selectively bind to NK cells, but do not bind to neutrophils. A multispecific killer engager compound that includes an NK engaging domain that includes an anti-CD16a antibody can bind to NK cells but not bind to neutrophils. Thus, in circumstances where one may want to engage NK cells but not engage neutrophils, one can design an NK engaging domain of the multispecific killer engager compound to include an anti-CD16a antibody.

In one or more embodiments, an immune cell engaging domain can involve the use of a humanized immune cell engager derived from an animal single domain antibody (sdAb) such as, for example, a humanized CD 16 engager derived from a sdAb. While an scFv has a heavy variable chain component and a light variable chain component joined by a linker, a nanobody consists of a single monomeric variable chain — i.e., a variable heavy chin or a variable light chain — that is capable of specifically engaging a target. A single domain antibody (sdAb) may be derived from an antibody of any suitable animal such as, for example, a camelid (e.g., a llama or camel) or a cartilaginous fish. A single domain antibody can provide superior physical stability, an ability to bind deep grooves, and increased production yields compared to larger antibody fragments.

In one exemplary embodiment, an sdAb-based NK engager molecule can involve a humanized CD16 nanobody derived from a llama nanobody (GeneBank sequence EF561291; Behar et al., 2008. Protein Eng Des Sei. 21(1): 1-10), termed EF91. Upon confirming functionality of the molecule, the CDRs were cloned into a humanized camelid scaffold (Vincke et al., 2009. J Biol Chem. 284(5):3273-3284) to humanize the CD16 engager (SEQ ID NO: 19). The use of a humanized camelid sdAb (e.g., SEQ ID NO:21) in the immune cell engaging domain of a multispecific killer engager compound can increase drug yield, increase stability, and/or increase NK-cell-mediated antibody-dependent cellular cytotoxicity (ADCC) efficacy.

While described herein in the context of various embodiments in which the immune cell engaging domain includes an anti-CD16 sdAb (e.g., SEQ ID NOs: l-5) or anti-CD16 scFv (e.g., SEQ ID NO:25), the immune cell engaging domain can include any antibody or other ligand that selectively binds to the immune cell or immune cells that the compound is designed to engage. Moreover, the immune cell engaging domain can include an antibody or ligand that selectively binds to any immune cell receptor. When the immune cell engaging domain is designed to engage NK cells, the NK engaging domain can selectively bind to, for example, the cell cytotoxicity receptor 2B4, low affinity Fc receptor CD 16, killer immunoglobulin like receptors (KIR), CD2, NKG2A, TIGIT, NKG2C, NKG2D, NKp44, NKp46, NKp30, LIR-1, and/or DNAM-1. In embodiments, in which the immune cell engaging domain is designed to engage T cells, the T cell engaging domain can selectively bind to CD3.

In one or more embodiments, a multispecific anti-TEM8 compound can including one or more additional immune cell engaging domains. In exemplary embodiments in which the multispecific anti-TEM8 compound is designed to engage NK cells, an additional NK engaging domain can specifically bind to any NK cell marker so that the compound, as a while, engages any combination of two or more NK cell markers. In one or more alternative embodiments, the immune cell engaging domains can engage different immune cells. Thus, one immune cell engaging domain may engage CD 16 on NK cells and a second immune cell engaging domain may engage a marker on a different immune population such as, for example, a monocyte marker, a macrophage marker, or a T cell marker. In one or more embodiments, a multispecific anti-TEM8 compound can include an immune cell activating domain. In one or more embodiments, the immune cell can be an NK cell and the immune cell activating domain includes a NK activating cytokine or a functional portion thereof.

An NK activating domain can include an amino acid sequence that activates NK cells, promotes sustaining NK cells, or otherwise promotes NK cell activity. For example, NK cells are responsive to a variety of cytokines including, but not limited to, IL- 15, which is involved in NK cell homeostasis, proliferation, survival, activation, and/or development. IL-15 and IL-2 share several signaling components, including the IL-2/IL-15RP (CD 122) and the common gamma chain (CD132). Unlike IL-2, IL-15 does not stimulate Tregs, allowing for NK cell activation while bypassing Treg inhibition of the immune response. Besides promoting NK cell homeostasis and proliferation, IL-15 can rescue NK cell functional defects that can occur in the post-transplant setting. IL- 15 also can stimulate CD8 ⁺ T cell function, further enhancing its immunotherapeutic potential. In addition, based on pre-clinical studies, toxicity profiles of IL- 15 may be more favorable than IL-2 at low doses.

Therefore, an NK activating domain can be, or can be derived from, one or more cytokines that can activate and/or sustain NK cells. As used herein, the term “derived from” refers to an amino acid fragment of a cytokine (e.g., IL-15) that is sufficient to provide NK cell activating and/or sustaining activity. In embodiments that include more than one NK activating domain, the NK activating domains may be provided in series or in any other combination. Additionally, each cytokine-based NK activating domain can include either the full amino acid sequence of the cytokine or may be an amino acid fragment, independent of the nature of other NK activating domains included in the multispecific killer engager compound. Exemplary cytokines on which an NK activating domain may be based include, for example, IL-15, IL-18, IL- 12, and IL-21.

Thus, while described in detail herein in the context of an exemplary model embodiment in which an NK activating domain is derived from IL-15, a multispecific anti-TEM8 compound may be designed using an NK activating domain that is, or is derived from, any suitable cytokine.

Moreover, while described herein in the context of exemplary embodiments in which the immune cell activating domain activates NK cells, a multispecific anti-TEM8 compound can be designed to have an immune cell activating domain that activates any desired population or subpopulation of immune cells.

For brevity in this description, reference to an immune cell activating domain by identifying the cytokine on which it is based includes both the full amino acid sequence of the cytokine, any suitable amino acid fragment of the cytokine, and or a modified version of the cytokine that includes one or more amino acid substitutions. Thus, reference to an “IL- 15” NK activating domain includes an NK activating domain that includes the full amino acid sequence of IL- 15, an NK activating domain that includes a fragment of IL- 15 (e.g., SEQ ID NO:22), a functional variant thereof, or an NK activating domain that includes an amino acid substitution compared to the wild-type IL-15 amino acid sequence. For example, an NK activating domain can include a fragment of IL-15 that includes an N-to-D or an N-to-A amino acid substitution at position 72 of SEQ ID NO:22. Reference to position 72 of SEQ ID NO:22 merely refers to the location of the amino acid substitution regardless of the particular fragment of IL-15 that may be used as the NK activating domain. Thus, the NK activating domain can include a fragment of IL- 15 other than the fragment reflected in SEQ ID NO:22 and that fragment can have an N-to-D or an N-to-A amino acid substitution at the position of the alternative IL- 15 fragment that corresponds to position 72 of SEQ ID NO:22.

In one or more embodiments, an NK activating domain can include wild-type human IL- 12, or any variant thereof that includes an amino acid sequence that activates NK cells, promotes sustaining NK cells, rescues NK cell exhaustion, or otherwise promotes NK cell activity (e.g., degranulation or IFNY production). IL-12 includes two signaling subunits, IL-12A and IL-12B. The IL-12-based NK activating domain includes, or is derived from, human IL-12A and/or IL- 126. As used herein, the term “derived from” refers to an amino acid fragment of IL-12A and/or IL-12B that is sufficient to provide NK cell activating and/or sustaining activity (e.g., induce degranulation, induce IFNy secretion, and/or increase IFNy in exhausted NK cells). Thus, the IL- 12-based NK activating domain can include the amino acid sequence of human IL-12A (SEQ ID NO:23), human IL-12B (SEQ ID NO:24), or both. When the NK activating domain includes both IL-12A and IL-12B, these components can be present in any order. Further, when the NK activating domain includes both IL-12A and IL-12B, the NK activating domain can further include additional amino acids such as, for example, a linker or flanking sequence. Suitable flanking sequences that may be used to link IL- 12 subunits are described in detail below. In embodiments in which a multispecific anti-TEM8 compound is designed to activate T cells, the multispecific anti-TEM8 compound can generally include one or more T cell engaging domains, one or more T cell activating domains, and one or more targeting domain (that target, e.g., a tumor cell or virally-infected cell), and one or more T cell activating domains (e.g., IL-2 or other T cell enhancing cytokine, chemokine, and/or activating molecule), with each domain operably linked to the other domains.

A T cell engaging domain can include any moiety that binds to and/or activates a T cell and/or any moiety that blocks inhibition of a T cell. In one or more embodiments, the T cell engaging domain can include an amino acid sequence. In one or more embodiments, a T cell engaging domain can include an antibody or fragment thereof that selectively binds to a component of the surface of a T cell. In other embodiments, a T cell engaging domain can include a ligand or small molecule that selectively binds to a component of the surface of a T cell.

In one or more embodiments, a T cell engaging domain can selectively bind to a receptor at least partially located at the surface of a T cell. In certain embodiments, a T cell engaging domain can serve a function of binding a T cell and thereby bring a T cell into spatial proximity with a target to which the targeting domain selectively binds. In certain embodiments, a T cell engaging domain can selectively bind to a receptor that also activates a T cell and therefore also possess an activating function.

Thus, in one or more embodiments, a T cell engaging domain includes an antibody or other ligand that selectively binds to the CD3 receptor such as, for example, an anti-CD3 receptor scFv or humanized anti-CD3 single domain antibody (sdAb). In other embodiments, a T cell engaging domain can include an antibody or ligand that selectively binds to any T cell receptor such as, for example, an anti-CD4 antibody, an anti-CD8 antibody, an anti-LFA-1 antibody, an anti-LFA-2 antibody, an anti-CTLA4 antibody, an anti-TCR antibody, an anti- CD28 antibody, an anti-CD25 antibody, an anti-PDl antibody, PD-1L, B7-1, B7-2, MHC molecules, CD80, CD86, B7H, an anti-SLAM antibody, or an anti-BTLA antibody.

In one or more embodiments, an anti-TEM8 compound can include more than one immune cell activating domain. In embodiments that include more than one immune cell activating domain, the additional immune cell activating domain or domains may be provided in series or in any other combination. Each additional immune cell activating domain can be selected, independently of every other immune cell activating domain, to include an immune cell activating cytokine. Further, each immune cell activating domain can include either the full amino acid sequence of the cytokine or an immune cell activating fragment of the cytokine, independent of the nature of other immune cell activating domains included in the anti-TEM8 compound.

In one or more embodiments, the anti-TEM8 compound includes domains operably linked by one or more linkers or flanking sequences. As used herein, the term “linker” refers generically to any of the amino acid sequences identified as a “flanking sequence” (SEQ ID NO:8-20) or any non-amino acid chemical linkers. Thus, a linker or flanking sequence can include the amino acid sequence of any one of SEQ ID NOs:8-20. In embodiments in which the multispecific anti-TEM8 compound includes more than one linker or flanking sequence, the linker or flanking sequence selected to link any two domains may be selected independently of any other linker or flanking sequence selected to link any other two domains. In specific exemplary embodiments (e.g., SEQ ID NOs: 1-5), the immune cell engaging domain is linked to an immune cell activating domain using the flanking sequence of SEQ ID NO:8, while the immune cell activating domain is linked to the targeting domain using the flanking sequence of SEQ ID NO:9.

As used herein, the term “operably linked” refers to a direct or indirect covalent linking between the domains of the multispecific compound. Thus, two domains that are operably linked may be directly covalently coupled to one another. Conversely, two operably linked domains may be connected by mutual covalent linking to an intervening moiety (e.g., a flanking sequence or linker). Two domains may be considered operably linked if, for example, they are separated by the third domain, with or without one or more intervening flanking sequences.

Suitable flanking sequences of a multispecific anti-TEM8 compound can include natural linkers, empirical linkers, or a combination of natural and empirical linkers. Natural linkers are derived from multi-domain proteins and, therefore, include polypeptide sequences of amino acid residues that are naturally present between protein domains. Properties of natural linkers such as, for example, length, hydrophobicity, amino acid residues, and/or secondary structure can be exploited to confer desirable properties to a multi-domain compound that includes natural linkers connecting functional domains. The studies of linkers in natural multi-domain proteins have led to the generation of many empirical linkers with various sequences and conformations for the construction of recombinant fusion proteins. Empirical linkers can be classified in three types: flexible linkers, rigid linkers, and cleavable linkers. Flexible linkers can provide a certain degree of movement or interaction at the joined domains. Flexible linkers typically include small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids, which provide flexibility, and allow for mobility of the connected functional domains. Rigid linkers can successfully keep a fixed distance between domains to maintain their independent functions, which can provide efficient separation of the protein domains and/or sufficiently reduce interference between functional domains. Cleavable linkers can allow one to control release of functional domains in vivo. By taking advantage of unique in vivo processes, cleavable linkers can be cleaved under specific conditions such as the presence of reducing reagents or proteases. This type of linker can reduce steric hindrance, improve bioactivity, and/or achieve independent actions/metabolism of individual domains of recombinant fusion proteins after linker cleavage.

Exemplary flanking sequences include polypeptides having the amino acid sequences of SEQ ID NOs:8-20.

Thus, while described herein in the context of exemplary anti-TEM8 multispecific compounds as reflected in SEQ ID NOs:l-5, this disclosure further describes the design elements required for a person of ordinary skill in the art to design, make, and use an anti-TEM8 multispecific compound that employs alternative or additional immune cell engaging domains, alternative or additional NK activating domains, alternative or additional anti-TEM8 targeting domains, and/or additional targeting domains, and functional variants of structurally similar to a reference anti-TEM8 multispecific compound.

As used herein, a polypeptide is “structurally similar” to a reference polypeptide if the amino acid sequence of the polypeptide possesses a specified amount of identity compared to the reference polypeptide. Structural similarity of two polypeptides can be determined by aligning the residues of the two polypeptides to optimize the number of identical amino acids along the lengths of their sequences; gaps in either or both sequences are permitted in making the alignment in order to optimize the number of identical amino acids, although the amino acids in each sequence must nonetheless remain in their proper order. The reference polypeptide can be, but is not limited to, any one of SEQ ID NOs: 1-5. In one or more embodiments, the reference polypeptide can be a novel polypeptide designed to have one or more alternatives to an immune cell engaging domain, an NK activating domain, or an anti-TEM8 targeting domain compared to any one of SEQ ID NOs: 1-5, as explained in the preceding paragraph. A candidate polypeptide is the polypeptide being compared to the reference polypeptide. A candidate polypeptide can be isolated, for example, from an animal, or can be produced using recombinant techniques, or chemically or enzymatically synthesized.

A pair-wise comparison analysis of amino acid sequences can be carried out using, for example, the BESTFIT algorithm in the GCG package (version 10.2, Madison WI). Alternatively, proteins may be compared using the Blastp program of the BLAST 2 search algorithm, as described by Tatiana et al., (FEMS Microbiol Lett, 174, 247-250 (1999)), and available on the National Center for Biotechnology Information (NCBI) website. The default values for all BLAST 2 search parameters may be used, including matrix = BLOSUM62; open gap penalty = 11, extension gap penalty = 1, gap x dropoff = 50, expect = 10, wordsize = 3, and filter on.

In the comparison of two amino acid sequences, structural similarity may be referred to by percent “identity” or may be referred to by percent “similarity.” “Identity” refers to the presence of identical amino acids. “Similarity” refers to the presence of not only identical amino acids but also the presence of conservative substitutions. A conservative substitution for an amino acid in an anti-TEM8 polypeptide may be selected from other members of the class to which the amino acid belongs. For example, it is well-known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and hydrophilicity) can be substituted for another amino acid without altering the activity of a protein, particularly in regions of the protein that are not directly associated with biological activity. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine, and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Conservative substitutions include, for example, Lys for Arg and vice versa to maintain a positive charge; Glu for Asp and vice versa to maintain a negative charge; Ser for Thr so that a free -OH is maintained; and Gin for Asn to maintain a free -NH2. Likewise, biologically active analogs of a protein containing deletions or additions of one or more contiguous or noncontiguous amino acids that do not eliminate a functional activity of the protein are also contemplated.

Generally, portions of an anti-TEM8 multispecific compound outside of CDRs in any antibody-based domain are more amenable to variation while maintaining anti-TEM8 functionality — i.e., specifically binding to TEM8.

An anti-TEM8 polypeptide as described herein can include a protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence similarity to a reference anti-TEM8 compound (e.g., any one of SEQ ID NOs: l-5).

An anti-TEM8 polypeptide as described herein can include a protein with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference anti-TEM8 compound (e.g., any one of SEQ ID NOs: l-5).

Variants of the disclosed sequences also include proteins, or full-length protein, that contain substitutions, deletions, or insertions into the protein backbone, that would still leave at least about 70% homology to the original protein over the corresponding portion. A yet greater degree of departure from homology is allowed if like-amino acids, i.e., conservative amino acid substitutions, do not count as a change in the sequence. Examples of conservative substitutions involve amino acids that have the same or similar properties. Illustrative amino acid conservative substitutions include the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; valine to isoleucine to leucine.

In some aspects, an anti-TEM8 polypeptide can include additional sequences, such as, for example, amino acids appended to the C-terminal or N-terminal of the anti-TEM8 polypeptide. Such modifications can, for example, facilitate purification by trapping on columns, the use of antibodies, or facilitate recovery when expressed recombinantly in a microbe. Such tags include, for example, a histidine-rich tag that allows purification of proteins on nickel columns and/or a leader sequence that can traffic recombinantly-expressed protein to the membrane of the cell in which it is recombinantly expressed. Such gene modification techniques and suitable additional sequences are well known in the molecular biology arts. In one or more embodiments, the C- terminal and/or N-terminal modification may be cleaved from the anti-TEM8 polypeptide before being incorporated into, for example, a pharmaceutical composition. In other embodiments, retaining a C-terminal or N-terminal modification may be desired for a given application — i.e., to facilitate immobilization to a substrate.

In another aspect, this disclosure describes an isolated nucleic acid sequence that encodes any embodiment of an anti-TEM8 polypeptide as described herein. Given the amino acid sequence of any anti-TEM8 polypeptide, or multispecific anti-TEM8 protein that includes an anti-TEM8 polypeptide, a person of ordinary skill in the art can determine the full scope of polynucleotides that encode that amino acid sequence using conventional, routine methods.

As used herein, the term “nucleic acid” or “oligonucleotide” refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include but are not limited to genomic DNA, cDNA, mRNA, iRNA, miRNA, tRNA, ncRNA, rRNA, and recombinantly produced and chemically synthesized molecules such as aptamers, plasmids, antisense DNA strands, shRNA, ribozymes, nucleic acids conjugates, and oligonucleotides. A nucleic acid may be single-stranded, double-stranded, linear, or covalently circularly closed molecule. A nucleic acid can be isolated. The term “isolated nucleic acid” means, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, (iv) was synthesized, for example, by chemical synthesis, or (vi) extracted from a sample. A nucleic might be introduced — i.e., transfected — into cells. When RNA is used to transfect cells, the RNA may be modified by stabilizing modifications, capping, or polyadenylation.

As used herein “amplified DNA” or “PCR product” refers to an amplified fragment of DNA of defined size. Various techniques are available and well known in the art to detect PCR products. PCR product detection methods include, but are not restricted to, gel electrophoresis using agarose or polyacrylamide gel and adding ethidium bromide staining (a DNA intercalant), labeled probes (radioactive or non-radioactive labels, southern blotting), labeled deoxyribonucleotides (for the direct incorporation of radioactive or non-radioactive labels) or silver staining for the direct visualization of the amplified PCR products; restriction endonuclease digestion, that relies agarose or polyacrylamide gel or high-performance liquid chromatography (HPLC); dot blots, using the hybridization of the amplified DNA on specific labeled probes (radioactive or non-radioactive labels); high-pressure liquid chromatography using ultraviolet detection; electro-chemiluminescence coupled with voltage-initiated chemical reach on/photon detection; and direct sequencing using radioactive or fluorescently labeled deoxyribonucleotides for the determination of the precise order of nucleotides with a DNA fragment of interest, oligo ligation assay (OLA), PCR, qPCR, DNA sequencing, fluorescence, gel electrophoresis, magnetic beads, allele specific primer extension (ASPE) and/or direct hybridization.

Generally, nucleic acid can be extracted, isolated, amplified, or analyzed by a variety of techniques such as those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Woodbury, NY 2,028 pages (2012); or as described in U.S. Pat. 7,957,913; U.S. Pat. 7,776,616; U.S. Pat. 5,234,809; U.S. Pub. 2010/0285578; and U.S. Pub. 2002/0190663. Examples of nucleic acid analysis include, but are not limited to, sequencing and DNA-protein interaction. Sequencing may be by any method known in the art. DNA sequencing techniques include classic dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, and next generation sequencing methods such as sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, Illumina/Solexa sequencing, allele specific hybridization to a library of labeled oligonucleotide probes, sequencing by synthesis using allele specific hybridization to a library of labeled clones that is followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, and SOLiD sequencing. Separated molecules may be sequenced by sequential or single extension reactions using polymerases or ligases as well as by single or sequential differential hybridizations with libraries of probes.

In another aspect, this disclosure describes a host cell including any of the isolated nucleic acid sequences and/or anti-TEM8 polypeptides described herein. The nucleic acid constructs of the present disclosure may be introduced into a host cell to be altered, thus allowing expression of the chimeric protein within the cell, thereby generating a genetically engineered cell. A variety of methods are known in the art and suitable for introducing a nucleic acid into a cell, including viral and non-viral mediated techniques. Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion. Other methods of transfection include proprietary transfection reagents such as LIPOFECTAMINE (Thermo Fisher Scientific, Inc., Waltham, MA), HILYMAX (Dojindo Molecular Technologies, Inc., Rockville, MD), FUGENE (Promega Corp., Madison, WI), JETPEI (Polyplus Transfection, Illkirch, France), EFFECTENE (Qiagen, Hilden, Germany) and DreamFect (OZ Biosciences, Inc USA, San Diego, CA).

The nucleic acid constructs described herein may be introduced into a host cell to be altered, thus allowing expression within the cell of the polypeptide encoded by the nucleic acid. A variety of host cells are known in the art and suitable for protein expression. Examples of typical cell used for transfection and protein expression include, but are not limited to, a bacterial cell, a eukaryotic cell, a yeast cell, an insect cell, or a plant cell such as, for example, E. coll. Bacillus, Streptomyces, Pichia pasloris. Salmonella lyphimiirium. Drosophila S2, Spodoptera SJ9, CHO, CHO-DG44, expiCHO, COS (e g., COS-7), 3T3-F442A, HeLa, HUVEC, HUAEC, NIH 3T3, Jurkat, 293, 293H, 293F, or expi293.

An anti-TEM8 compound as described herein may be formulated with a pharmaceutically acceptable carrier. As used herein, “carrier” includes any solvent, dispersion medium, vehicle, coating, diluent, antibacterial, and/or antifungal agent, isotonic agent, absorption delaying agent, buffer, carrier solution, suspension, colloid, and the like. The use of such media and/or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with an anti-TEM8 compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

An anti-TEM8 compound may therefore be formulated into a pharmaceutical composition. The pharmaceutical composition may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A composition also can be administered via a sustained or delayed release.

Thus, an anti-TEM8 compound may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like.

A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing an anti-TEM8 compound into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and/or intimately bringing the active molecule into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

The amount of an anti-TEM8 compound administered can vary depending on various factors including, but not limited to, the particular anti-TEM8 compound being used, the weight, physical condition, and/or age of the subject, and/or the route of administration. Thus, the absolute weight of anti-TEM8 compound included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and/or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of anti-TEM8 compound effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.

In one or more embodiments, the method can include administering sufficient anti-TEM8 compound to provide a dose of, for example, from about 100 ng/kg to about 50 mg/kg to the subject, although in one or more embodiments the methods may be performed by administering the anti-TEM8 compound in a dose outside this range. In some of these embodiments, the method includes administering sufficient the anti-TEM8 compound to provide a dose of from about 10 pg/kg to about 5 mg/kg to the subject, for example, a dose of from about 100 pg/kg to about 1 mg/kg.

In another aspect, this disclosure describes a method including administering to a subject a multispecific compound in an amount effective to induce NK-mediated killing of a cell, the multispecific compound including a TEM8-targeting domain and an immune cell engaging domain operably linked to the TEM8-targeting domain. The TEM8-targeting domain generally includes any one of the anti-TEM8 polypeptides described herein.

In another aspect, this disclosure describes a method for stimulating expansion of NK cells in vivo including administering to a subject an amount of multispecific compound effective to stimulate expansion of NK cells in vivo, the multispecific compound including a TEM8- targeting domain and an immune cell engaging domain operably linked to the TEM8-targeting domain, The TEM8-targeting domain generally includes any one of the anti-TEM8 polypeptides described herein.

In another aspect, this disclosure describes methods of killing a target cell in a subject. Generally, the method includes administering to the subject an anti-TEM8 multispecific compound in an amount effective to induce NK-mediated killing of the target cells. “Treat” or variations thereof refer to reducing, limiting progression, ameliorating, or resolving, to any extent, the symptoms or signs related to a condition. As used herein, “ameliorate” refers to any reduction in the extent, severity, frequency, and/or likelihood of a symptom or clinical sign characteristic of a particular condition; “symptom” refers to any subjective evidence of disease or of a subject’s condition; and “sign” or “clinical sign” refers to an objective physical finding relating to a particular condition capable of being found by one other than the subject. A “treatment” may be therapeutic or prophylactic. “Therapeutic” and variations thereof refer to a treatment that ameliorates one or more existing symptoms or clinical signs associated with a condition. “Prophylactic” and variations thereof refer to a treatment that limits, to any extent, the development and/or appearance of a symptom or clinical sign of a condition. Generally, a “therapeutic” treatment is initiated after the condition manifests in a subject, while “prophylactic” treatment is initiated before a condition manifests in a subject. Thus, in certain embodiments, the method can involve prophylactic treatment of a subject at risk of developing a condition. “At risk” refers to a subject that may or may not actually possess the described risk. Thus, for example, a subject “at risk” for developing a specified condition is a subject that possesses one or more indicia of increased risk of having, or developing, the specified condition compared to individuals who lack the one or more indicia, regardless of the whether the subject manifests any symptom or clinical sign of having or developing the condition. Exemplary indicia of a condition can include, for example, genetic predisposition, ancestry, age, sex, geographical location, lifestyle, or medical history. Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

In some cases, the treatment can involve administering the anti-TEM8 multispecific compound to a subject so that the anti-TEM8 multispecific compound can stimulate endogenous NK cells in vivo. Using an anti-TEM8 multispecific compound as a part of an in vivo can make NK cells antigen specific with simultaneous co-stimulation, enhancement of survival, and expansion, which may be antigen specific. In other cases, the anti-TEM8 multispecific compound can be used in vitro as an adjuvant to NK cell adoptive transfer therapy. The terms “administration of’ and or “administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical, or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration. Accordingly, an anti-TEM8 multispecific compound may be administered before, during, or after the subject first exhibits a symptom or clinical sign of the condition. Treatment initiated before the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the likelihood that the subject experiences clinical evidence of the condition compared to a subject to which the anti-TEM8 multispecific compound is not administered, decreasing the severity of symptoms and/or clinical signs of the condition, and/or completely resolving the condition. Treatment initiated after the subject first exhibits a symptom or clinical sign associated with the condition may result in decreasing the severity of symptoms and/or clinical signs of the condition compared to a subject to which the composition is not administered, and/or completely resolving the condition.

The anti-TEM8 multispecific compound can be any embodiment of the anti-TEM8 multispecific compound described above having a targeting domain that selectively binds to an appropriate target cell population. In some cases, the target cell can include a tumor cell so that the method can involve treating cancer associated with the tumor cells. Thus, in one or more embodiments, the method can include ameliorating at least one symptom or clinical sign of the tumor.

In embodiments in which the target cell includes a tumor cell, the method can further include surgically resecting the tumor and/or reducing the size of the tumor through chemical (e.g., chemotherapeutic) and/or radiation therapy. Exemplary tumors that may be treated include tumors associated with prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer and/or hematopoietic cancer.

Thus, in one or more embodiments, treating a subject includes a subject having, or at risk of having cancer. Generally, the method includes administering to the subject an effective amount of multispecific compound including a targeting domain including one of the anti-TEM8 polypeptides described herein and an immune cell engaging domain operably linked to the anti- TEM8 polypeptide. As used herein, the term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation starting at one site (primary site) with the potential to invade and to spread to other sites (secondary sites, metastases) which differentiates cancer (malignant tumor) from benign tumor. Virtually any organ can be affected, meaning more than 100 types of cancer can affect humans. Cancers can result from many causes including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to an environmental pollutant, tobacco and/or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, “neoplasm” or “tumor” (and grammatical variations thereof) means new and abnormal growth of tissue, which may be benign or cancerous. In a related aspect, the neoplasm is indicative of a neoplastic disease or disorder, including but not limited, to various cancers. For example, such cancers can include prostate, pancreatic, biliary, colon, rectal, liver, kidney, lung, testicular, breast, ovarian, brain, and head and neck cancers, melanoma, sarcoma, multiple myeloma, leukemia, lymphoma, and the like.

Exemplary cancers described by the national cancer institute include: Acute Lymphoblastic Leukemia, Adult; Acute Lymphoblastic Leukemia, Childhood; Acute Myeloid Leukemia, Adult; Adrenocortical Carcinoma; Adrenocortical Carcinoma, Childhood; AIDS- Related Lymphoma; AIDS-Related Malignancies; Anal Cancer; Astrocytoma, Childhood Cerebellar; Astrocytoma, Childhood Cerebral; Bile Duct Cancer, Extrahepatic; Bladder Cancer; Bladder Cancer, Childhood; Bone Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma; Brain Stem Glioma, Childhood; Brain Tumor, Adult; Brain Tumor, Brain Stem Glioma, Childhood; Brain Tumor, Cerebellar Astrocytoma, Childhood; Brain Tumor, Cerebral Astrocytoma/Malignant Glioma, Childhood; Brain Tumor, Ependymoma, Childhood; Brain Tumor, Medulloblastoma, Childhood; Brain Tumor, Supratentorial Primitive Neuroectodermal Tumors, Childhood; Brain Tumor, Visual Pathway and Hypothalamic Glioma, Childhood; Brain Tumor, Childhood (Other); Breast Cancer; Breast Cancer and Pregnancy; Breast Cancer, Childhood; Breast Cancer, Male; Bronchial Adenomas/Carcinoids, Childhood: Carcinoid Tumor, Childhood; Carcinoid Tumor, Gastrointestinal; Carcinoma, Adrenocortical; Carcinoma, Islet Cell; Carcinoma of Unknown Primary; Central Nervous System Lymphoma, Primary; Cerebellar Astrocytoma, Childhood; Cerebral Astrocytoma/Malignant Glioma, Childhood; Cervical Cancer; Childhood Cancers; Chronic Lymphocytic Leukemia; Chronic Myelogenous Leukemia; Chronic Myeloproliferative Disorders; Clear Cell Sarcoma of Tendon Sheaths; Colon Cancer; Colorectal Cancer, Childhood; Cutaneous T-Cell Lymphoma; Endometrial Cancer; Ependymoma, Childhood; Epithelial Cancer, Ovarian; Esophageal Cancer; Esophageal Cancer, Childhood; Ewing’s Family of Tumors; Extracranial Germ Cell Tumor, Childhood; Extragonadal Germ Cell Tumor; Extrahepatic Bile Duct Cancer; Eye Cancer, Intraocular Melanoma; Eye Cancer, Retinoblastoma; Gallbladder Cancer; Gastric (Stomach) Cancer; Gastric (Stomach) Cancer, Childhood; Gastrointestinal Carcinoid Tumor; Germ Cell Tumor, Extracranial, Childhood; Germ Cell Tumor, Extragonadal; Germ Cell Tumor, Ovarian; Gestational Trophoblastic Tumor; Glioma. Childhood Brain Stem; Glioma. Childhood Visual Pathway and Hypothalamic; Hairy Cell Leukemia; Head and Neck Cancer; Hepatocellular (Liver) Cancer, Adult (Primary);

Hepatocellular (Liver) Cancer, Childhood (Primary); Hodgkin’s Lymphoma, Adult; Hodgkin’s Lymphoma, Childhood; Hodgkin’s Lymphoma During Pregnancy; Hypopharyngeal Cancer; Hypothalamic and Visual Pathway Glioma, Childhood; Intraocular Melanoma; Islet Cell Carcinoma (Endocrine Pancreas); Kaposi’s Sarcoma; Kidney Cancer; Laryngeal Cancer; Laryngeal Cancer, Childhood; Leukemia, Acute Lymphoblastic, Adult; Leukemia, Acute Lymphoblastic, Childhood; Leukemia, Acute Myeloid, Adult; Leukemia, Acute Myeloid, Childhood; Leukemia, Chronic Lymphocytic; Leukemia, Chronic Myelogenous; Leukemia, Hairy Cell; Lip and Oral Cavity Cancer; Liver Cancer, Adult (Primary); Liver Cancer, Childhood (Primary); Lung Cancer, Non-Small Cell; Lung Cancer, Small Cell; Lymphoblastic Leukemia, Adult Acute; Lymphoblastic Leukemia, Childhood Acute; Lymphocytic Leukemia, Chronic; Lymphoma, AIDS — Related; Lymphoma, Central Nervous System (Primary);

Lymphoma, Cutaneous T-Cell; Lymphoma, Hodgkin’s, Adult; Lymphoma, Hodgkin’s; Childhood; Lymphoma, Hodgkin’s During Pregnancy; Lymphoma, Non-Hodgkin’s, Adult; Lymphoma, Non-Hodgkin’s, Childhood; Lymphoma, Non-Hodgkin’s During Pregnancy; Lymphoma, Primary Central Nervous System; Macroglobulinemia, Waldenstrom’s; Male Breast Cancer; Malignant Mesothelioma, Adult; Malignant Mesothelioma, Childhood; Malignant Thymoma; Medulloblastoma, Childhood; Melanoma; Melanoma, Intraocular; Merkel Cell Carcinoma; Mesothelioma, Malignant; Metastatic Squamous Neck Cancer with Occult Primary; Multiple Endocrine Neoplasia Syndrome, Childhood; Multiple Myeloma/Plasma Cell Neoplasm; Mycosis Fungoides; Myelodysplasia Syndromes; Myelogenous Leukemia, Chronic; Myeloid Leukemia, Childhood Acute; Myeloma, Multiple; Myeloproliferative Disorders, Chronic; Nasal Cavity and Paranasal Sinus Cancer; Nasopharyngeal Cancer; Nasopharyngeal Cancer, Childhood; Neuroblastoma; Non-Hodgkin’s Lymphoma, Adult; Non-Hodgkin’s Lymphoma, Childhood; Non-Hodgkin’s Lymphoma During Pregnancy; Non-Small Cell Lung Cancer; Oral Cancer, Childhood; Oral Cavity and Lip Cancer; Oropharyngeal Cancer;

Osteosarcoma/Malignant Fibrous Histiocytoma of Bone; Ovarian Cancer, Childhood; Ovarian Epithelial Cancer; Ovarian Germ Cell Tumor; Ovarian Low Malignant Potential Tumor; Pancreatic Cancer; Pancreatic Cancer, Childhood, Pancreatic Cancer, Islet Cell; Paranasal Sinus and Nasal Cavity Cancer; Parathyroid Cancer; Penile Cancer; Pheochromocytoma; Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood; Pituitary Tumor; Plasma Cell Neoplasm/Multiple Myeloma; Pleuropulmonary Blastoma; Pregnancy and Breast Cancer; Pregnancy and Hodgkin’s Lymphoma; Pregnancy and Non-Hodgkin’s Lymphoma; Primary Central Nervous System Lymphoma; Primary Liver Cancer, Adult; Primary Liver Cancer, Childhood; Prostate Cancer; Rectal Cancer; Renal Cell (Kidney) Cancer; Renal Cell Cancer, Childhood; Renal Pelvis and Ureter, Transitional Cell Cancer; Retinoblastoma;

Rhabdomyosarcoma, Childhood; Salivary Gland Cancer; Salivary Gland’s Cancer, Childhood; Sarcoma, Ewing's Family of Tumors; Sarcoma, Kaposi’s; Sarcoma (Osteosarcoma) Malignant Fibrous Histiocytoma of Bone; Sarcoma, Rhabdomyosarcoma, Childhood; Sarcoma, Soft Tissue, Adult; Sarcoma, Soft Tissue, Childhood; Sezary Syndrome; Skin Cancer; Skin Cancer, Childhood; Skin Cancer (Melanoma); Skin Carcinoma, Merkel Cell; Small Cell Lung Cancer; Small Intestine Cancer; Soft Tissue Sarcoma, Adult; Soft Tissue Sarcoma, Childhood; Squamous Neck Cancer with Occult Primary, Metastatic; Stomach (Gastric) Cancer; Stomach (Gastric) Cancer, Childhood; Supratentorial Primitive Neuroectodermal Tumors, Childhood; T-Cell Lymphoma, Cutaneous; Testicular Cancer; Thymoma, Childhood; Thymoma, Malignant; Thyroid Cancer; Thyroid Cancer, Childhood; Transitional Cell Cancer of the Renal Pelvis and Ureter; Trophoblastic Tumor, Gestational; Unknown Primary Site, Cancer of, Childhood; Unusual Cancers of Childhood; Ureter and Renal Pelvis, Transitional Cell Cancer; Urethral Cancer; Uterine Sarcoma; Vaginal Cancer; Visual Pathway and Hypothalamic Glioma, Childhood; Vulvar Cancer; Waldenstrom’s Macro globulinemia; and Wilms’ Tumor.

In one or more embodiments, the cancer can include or involve prostate cancer, lung cancer, colon cancer, rectum cancer, urinary bladder cancer, melanoma, kidney cancer, renal cancer, oral cavity cancer, pharynx cancer, pancreas cancer, uterine cancer, thyroid cancer, skin cancer, head and neck cancer, cervical cancer, ovarian cancer, and/or hematopoietic cancer.

In one aspect, the multispecific compound is administered prior to, simultaneously with, or following chemotherapy, surgical resection of a tumor, or radiation therapy.

In one or more embodiments, a multispecific anti-TEM8 compound may be administered, for example, from a single dose to multiple doses per week, although in one or more embodiments the method can be performed by administering a multispecific anti-TEM8 compound at a frequency outside this range. In certain embodiments, a multispecific anti-TEM8 compound may be administered from about once per month to about five times per week.

In one or more embodiments, the method further includes administering one or more additional therapeutic agents. The one or more additional therapeutic agents may be administered before, after, and/or coincident to the administration of a multispecific anti-TEM8 compound. A multispecific anti-TEM8 compound and the additional therapeutic agents may be coadministered. As used herein, “co-administered” refers to two or more components of a combination administered so that the therapeutic or prophylactic effects of the combination can be greater than the therapeutic or prophylactic effects of either component administered alone. Two components may be co-administered simultaneously or sequentially. Simultaneously coadministered components may be provided in one or more pharmaceutical compositions. Sequential co-administration of two or more components includes cases in which the components are administered so that each component can be present at the treatment site at the same time. Alternatively, sequential co-administration of two components can include cases in which at least one component has been cleared from a treatment site, but at least one cellular effect of administering the component (e.g., cytokine production, activation of a certain cell population, etc.) persists at the treatment site until one or more additional components are administered to the treatment site. Thus, a co-administered combination can, in certain circumstances, include components that never exist in a chemical mixture with one another. In other embodiments, the multispecific anti-TEM8 compound and the additional therapeutic agent may be administered as part of a mixture or cocktail. In some aspects, the administration of the multispecific anti-TEM8 compound may allow for the effectiveness of a lower dosage of other therapeutic modalities when compared to the administration of the other therapeutic agent or agents alone, thereby decreasing the likelihood, severity, and/or extent of the toxicity observed when a higher dose of the other therapeutic agent or agents is administered.

The term “chemotherapeutic agent” as used herein refers to any therapeutic agent used to treat cancer. Examples of chemotherapeutic agents include, but are not limited to, actinomycin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed, teniposide, tioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, panitumamab, Erbitux™ (cetuximab), matuzumab, IMC-IIF 8, TheraCIM hR3, denosumab, Avastin™ (bevacizumab), Humira™ (adalimumab), Herceptin™ (trastuzumab), Remicade™ (infliximab), rituximab, Synagis™ (palivizumab), Mylotarg™ (gemtuzumab oxogamicin), Raptiva™ (efalizumab), Tysabri™ (natalizumab), Zenapax™ (dacliximab), NeutroSpec™ (Technetium (99mTc) fanolesomab), tocilizumab, ProstaScint™ (Indium-Ill labeled Capromab Pendetide), Bexxar™ (tositumomab), Zevalin™ (ibritumomab tiuxetan (IDEC-Y2B8) conjugated to yttrium 90), Xolair™ (omalizumab), Mab Thera™ (Rituximab), ReoPro™ (abciximab), MabCampath™ (alemtuzumab), Simulect™ (basiliximab), LeukoScan™ (sulesomab), CEA-Scan™ (arcitumomab), Verluma™ (nofetumomab), Panorex™ (Edrecolomab), alemtuzumab, CDP 870, natalizumab Gilotrif™ (afatinib), Lynparza™ (olaparib), Perjeta™ (pertuzumab), Otdivo™ (nivolumab), Bosulif™ (bosutinib), Cabometyx™ (cabozantinib), Ogivri™ (trastuzumab-dkst), Sutent™ (sunitinib malate), Adcetris™ (brentuximab vedotin), Alecensa™ (alectinib), Calquence™ (acalabrutinib), Yescarta™ (ciloleucel), Verzenio™ (abemaciclib), Keytruda™ (pembrolizumab), Aliqopa™ (copanlisib), Nerlynx™ (neratinib), Imfinzi™ (durvalumab), Darzalex™ (daratumumab), Tecentriq™ (atezolizumab), and Tarceva™ (erlotinib). Examples of immunotherapeutic agent include, but are not limited to, interleukins (11-2, 11-7, 11-12), cytokines (Interferons, G-CSF, imiquimod), chemokines (CCL3, CC126, CXCL7), immunomodulatory imide drugs (thalidomide and its analogues).

In some aspects, the chemotherapy is selected from the group consisting of altretamine, amsacrine, L-asparaginase, colaspase, bleomycin, busulfan, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytophosphane, cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, etoposide, fluorouracil, fludarabine, fotemustine, ganciclovir, gemcitabine, hydroxyurea, idarubicin, ifosfamaide, irinotecan, lomustine, melphalan, mercaptopurine, methotrexate, mitoxantrone, mitomycin C, nimustine, oxaliplatin, paclitaxel, pemetrexed, procarbazine, raltitrexed, temozolomide, teniposide, tioguanine, thiotepa, topotecan, vinblastine, vincristine, vindesine, and vinorelbine. In one or more embodiments, the method can include administering sufficient multispecific anti-TEM8 compound as described herein and administering the at least one additional therapeutic agent demonstrates therapeutic synergy. In some aspects of the methods of the present invention, a measurement of response to treatment observed after administering both a multispecific anti-TEM8 compound as described herein and the additional therapeutic agent is improved over the same measurement of response to treatment observed after administering either the multispecific anti-TEM8 compound or the additional therapeutic agent alone.

The term “subject” as used herein refers to any individual or subject to which the subject methods are performed. In many embodiments, the subject is human, although the subject may be any non-human animal. Suitable non-human animals include, but are limited to, a vertebrate such as a rodent (including a mouse, a rat, a hamster, or a guinea pig), a cat, a dog, a rabbit, a farm animal (including a cow, a horse, a goat, a sheep, a pig, a chicken, etc.), or a primate (including a monkey, a chimpanzee, an orangutan, or a gorilla).

In one or more embodiments of this aspect, the anti-TEM8 multispecific compound can include an immune cell activating domain that includes IL-15 or a functional portion thereof, operably linked to the immune cell engaging domain. In one or more embodiments, the multispecific anti-TEM8 compound can have the amino acid sequence as set forth in any one of SEQ ID NOs:20-25.

In another aspect, this disclosure describes a chimeric antigen receptor compound that includes one of the anti-TEM8 polypeptides described herein. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors, or artificial T cell receptors) are receptor proteins that have been engineered to give T cells the new ability to target a specific protein. The receptors are chimeric because they combine both antigen binding and T cell activating functions into a single receptor.

CAR-T cell therapy uses T cells engineered with CARs for cancer therapy. The premise of CAR-T immunotherapy is to modify T cells to recognize cancer cells to target and destroy the cancer cells more effectively. T cells are harvested from a donor (autologous or allogeneic), genetically altered, then the resulting CAR-T cells are infused into a subject to attack the subject’s tumor. CAR-T cells can be either derived from T cells in a subject’s own blood (autologous) or derived from the T cells of a donor (allogeneic). Once isolated from a person, these T cells are genetically engineered to express a specific CAR that programs the T cells to target an antigen that is present on the surface of a tumor. For safety, CAR-T cells are engineered to be specific to an antigen expressed on a tumor that is not expressed on healthy cells. After CAR-T cells are infused into a subject, they act as a “living drug” against cancer cells. When the CAR-T cells contact their targeted antigen on a cell, CAR-T cells bind to the antigen, become activated, proliferate, and become cytotoxic. CAR-T cells destroy cells through several mechanisms including extensive stimulated cell proliferation, increasing the degree to which they are toxic to other living cells (cytotoxicity), and by inducing increased secretion of factors that can affect other cells (e.g., cytokines, interleukins, and/or growth factors).

In another aspect, this disclosure describes a targeted therapeutic compound that includes a targeting domain and a therapeutic domain linked to the targeting domain. The targeting domain includes any embodiment of the anti-TEM8 polypeptides described herein. In one or more embodiments, the targeted therapeutic compound can provide immunotherapy and, therefore, be a targeted immunotherapeutic compound. In one or more embodiments, the therapeutic domain can include a drug, a therapeutic radioisotope, a toxin, a cytokine, or a chemokine.

As used herein, the term “drug” refers to any chemical substance, which, when administered to a living organism, produces a biological effect. A pharmaceutical drug is a chemical substance used to treat, cure, prevent, or diagnose a disease or to promote well-being. Drugs can be obtained through extraction from medicinal plants, or by organic synthesis. Pharmaceutical drugs may be used for a limited duration, or on a regular basis for chronic disorders.

A “radioisotope” or “radionuclide” is an atom that has excess nuclear energy, making it unstable. This excess energy can be used in one of three ways: emitted from the nucleus as gamma radiation; transferred to one of its electrons to release it as a conversion electron; or used to create and emit a new particle (alpha particle or beta particle) from the nucleus. During those processes, the radionuclide is said to undergo radioactive decay. These emissions are considered ionizing radiation because they are powerful enough to liberate an electron from another atom. The radioactive decay can produce a stable nuclide or will sometimes produce a new unstable radionuclide that may undergo further decay.

As used herein, the term “toxin” refers to a substance harmful to cells. Toxins can be small molecules, peptides, or proteins that are capable of causing disease or cell death on contact with, or absorption by, body tissues. Toxins vary greatly in their toxicity. Toxins are largely secondary metabolites, which are organic compounds that are not directly involved in an organism’s growth, development, or reproduction, instead often aiding the organism in matters of defense. In some applications, a toxin may be used therapeutically by targeting the effect of the toxin toward an undesirable cell or cells (e.g., tumor cells).

Cytokines are a broad category of small proteins (-5-20 kDa) involved in cell signaling. Cytokines are peptides and cannot cross the lipid bilayer of cells to enter the cytoplasm, but are nevertheless involved in autocrine, paracrine, and endocrine signaling as immunomodulating agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in the terminology). Cytokines are produced by a broad range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, mast cells, endothelial cells, fibroblasts, and various stromal cells. Cytokines modulate the balance between humoral and cell-based immune responses, and they regulate the maturation, growth, and responsiveness of particular cell populations.

In a further aspect, this disclosure describes a targeted imaging compound that includes a targeting domain and an imaging domain linked to the targeting domain. The targeting domain includes any embodiment of one of the anti-TEM8 polypeptides described herein. The imaging domain can include any moiety that can produce a detectable signal. Exemplary imaging moieties include, but are not limited to a colorimetric label, a fluorescent label, a radioactive label, a magnetic label, or an enzymatic label.

In one or more embodiments, the anti-TEM8 polypeptides can be used in the context of a diagnostic assay or device. In one or more of these embodiments, an anti-TEM8 polypeptide can include a detectable label that allows detection of the compound after the compound is administered to a subject or is contacted with a sample that contains TEM8 to which the anti- TEM8 polypeptide binds. The detectable label can be any suitable detectable label. Exemplary detectable labels include, but are not limited to, a radioactive label, a fluorescent label, an enzymatic label, a colorimetric label, a magnetic label, and the like.

In one or more embodiments, an anti-TEM8 polypeptide may be immobilized to a substrate to, for example, capture TEM8 that is present in a sample. Captured TEM8 may be detected using any suitable method for detecting captured ligands. Alternatively, captured TEM8 may be released from the anti-TEM8 polypeptide and collected using any suitable method for release and collection of captured ligands.

In the preceding description and following claims, the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention. The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES caml615TEM8 Plasmid and Protein Production

A hybrid gene encoding the caml615TEM8 multispecific anti-TEM8 compound (SEQ ID NO:25) was cloned into the Minicircle plasmid pMC.EFla-MCS-SV40polyA (System Biosciences, LLC, Palo alto, CA). The hybrid gene encoded a camelid anti-CD16 single domain antibody (amino acids 1-122 of SEQ ID NO:25; Felices et al., Cancer Immunol Res 8: 1139- 1149, 2020), a (SGGGG) ₄ SG linker (SEQ ID NO:8; amino acids 123-144 of SEQ ID NO:25), an IL-15 moiety (amino acids 145-258 of SEQ ID NO:25), Whitlow linker (SEQ ID NO:9; amino acids 259-276 of SEQ ID NO:25), and an anti-TEM8 scFv (amino acids 277-515 of SEQ ID NO:25). The protein encoded by the hybrid gene also includes a 10X histidine tag. DNA sequencing analysis confirmed the final plasmid (Genomics Center, University of Minnesota, Minneapolis, MN). The caml615TEM8-encoding plasmid was transfected into Expi293F cells (Thermo Fisher Scientific, Inc., Waltham, MA). caml615TEM8 protein was isolated with the AKTA Avant chromatography system (GE Healthcare, Chicago, IL). Protein purity and size was determined by polyacrylamide gel electrophoresis and densitometry using ImageJl (Schneider et al., Nature Methods 9(7):671-675). An anti-TEM8 single chain variable fragment (scFv) without the anti-CD16 camelid nanobody and IL- 15 moiety was produced in the same manner.

Peripheral Blood Mononuclear Cell Donors, NK Cell Enrichment, and NK cell Expansion

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of de- identified healthy donors obtained by Memorial Blood Bank (Minneapolis, MN) using densitygradient centrifugation with Ficoll-Paque Premium (GE Healthcare, Chicago, IL). Whole PBMCs were used directly (“fresh”) or were cryopreserved and thawed one day before use. NK cells were enriched using an NK cell enrichment kit (STEMCELL Technologies, Inc., Vancouver, BC, Canada). For in vivo experiments, enriched NKs were expanded ex vivo as previously described (Somanchi et al., J Vis Exp 48:e2540, 2011), with minor modifications. During the two weeks of expansion, NKs were expanded in G-REX 6 well plates (Wilson Wolf Corp., New Brighton, MN). The irradiated K562 mIL21 41BBL cells were generated and used as previously described (Zhu et al., Blood 135(6):399-410).

Cell Lines and Cell Culture All cell lines and culture conditions are outlined in supplementary Table 1.

Table 1. Cell lines and cell culture

¹ DMEM and RPMI 1640 medias were supplemented with 10% Fetal Bovine Serum (FBS), while MEM a nucleosides was supplemented with 0.1 mM 2-mercaptoethanol, 500 U/mL recombinant human IL-2, 12.5% horse serum, and 12.5% FBS.

ANTXR1 expression data for cell lines was obtained from the DepMap 20Q1 Public dataset (Ghandi et al., Nature 569(7757):503-508, 2019). A549s transfected with green fluorescent protein and luciferase — hereafter A549/GFP/Luc — were obtained from Dan Vallera at the University of Minnesota. NucLight Red-labeled tumor cell lines were generated using IncuCyte NucLight Red lentivirus reagent (Sartorius, Gottingen, Germany).

Generation of TEM8 knockout A549 cell line

A CRISPR/Cas9 system was designed to knock out exon 1 of the ANTXR1 gene (NCBI Gene ID 84168) in the A549 cell line. Guide RNAs flanking the first exon were designed using a custom Cas9 crRNA design tool (CUSTOM ALT-R, Integrated DNA Technologies, Inc., Coralville, IA). The upstream crRNA spacer sequence was 5'- AATCTGGGACAAAGAACCGT-3' (SEQ ID NO:6), and the downstream crRNA spacer sequence was 5'- GCCTTTCCCACCAACACGGG-3' (SEQ ID NO: 7). crRNAs, tracrRNA, and Cas9 nuclease (ALT-R EUFI Cas9 Nuclease V3, Integrated DNA Technologies, Inc., Coralville, IA) were transfected into A549 cells using a transfection agent (LIPOFECTAMINE CRISPRMAX, Invitrogen, Carlsbad, CA. The knockout status of single cell clones was determined using polymerase chain reaction of genomic DNA.

Antibodies and Fluorescent Stains

Antibodies and flow cytometry reagents are listed in Table 2.

Table 2. Antibodies and Cytometry Reagents

NK Cell Degranulation and Cytokine Production Assay

Fresh or frozen PBMCs were co-cultured for five hours with target cells (5: 1 effectortarget) and caml615TEM8 serially diluted 1 :3 from 9 nM to 1.37 pM (used in all serial dilutions unless otherwise noted). anti-CD107a was added to detect NK cell degranulation as previously described (Bryceson et al., J Exp Med 202(7): 1001-1012, 2005). GOLGIPLUG (BD Biosciences, San Jose, CA) and GOLGISTOP (BD Biosciences, San Jose, CA) were added for the last four hours of co-culture. Cells were stained with the Live/Dead Fixable Near IR, anti- CD56, and anti-CD3, fixed, permeabilized, and stained with anti-fFNy. All flow-based assays were run on a flow cytometer (LSRII, BD Biosciences, San Jose, CA) and analyzed with FLOWJO software (BD Biosciences, San Jose, CA). TEM8 blocking assays were performed similarly except target cells were first incubated for 45 minutes in 1800 nM anti-TEM8 scFv before adding PBMCs and 9 nM caml615TEM8.

Tumor Spheroid Killing Assay

20,000 NucLight Red-expressing target cells were added per well to a round bottom ultra-low adhesion 96-well plate (Coming, Inc., Corning, NY). Spheroids were allowed to form for five days before 100,000 NK cells enriched from fresh PBMCs were added with 3 nM caml615TEM8 or 3 nM IL-15 for six days within a cell imager (INCUCYTE S3, Essen Biosciences, Inc., Ann Arbor, MI). The INCUCYTE S3 software was used to calculate red fluorescence area as a measure of tumor size. NK cells from four donors were tested, and each treatment was run in quintuplicate.

NK Cell and T Cell Proliferation Assay

PBMCs were stained with a cell proliferation kit (CELL TRACE, Thermo Fisher Scientific, Inc., Waltham, MA) and incubated for seven days with IL-15 or caml615TEM8 serially diluted as noted. PBMCs were then stained with Live/Dead Fixable Near IR, Annexin V, anti-CD56, and anti-CD3. “Proliferated” cells divided at least once, while “highly proliferated” cells divided at least two times.

NK Cell and T cell pSTAT5 Induction Assay

PBMCs were incubated for 20 minutes with IL- 15 or caml615TEM8 serially diluted as noted. PBMCs were then stained with anti-CD56 and anti-CD3, fixed, permeabilized with a permeabilization buffer (TRUE-PHOS, BioLegend, San Diego, CA), and stained with anti- STAT5/pY694. pSTAT5 levels in CD56 ⁺ CD3" NK cells and CD56' CD3 ⁺ T cells were measured by flow cytometry.

NK-92 Metabolic Activity Assay NK-92s were grown in IL-2 free media for 24 hours before the start of the assay. At the start of the assay, 50,000 viable NK-92s or NK-92/CD16s were plated per well in a clear bottom 96-well plate (Thermo Fisher Scientific, Inc., Waltham, MA) with IL-15 or caml615TEM8 serially diluted 1 : 10 seven times to generate dose-response curves for each drug. The highest concentration of IL- 15 was 150 nM while the highest concentration of caml615TEM8 was 1,500 nM. Each treatment concentration was run in triplicate. After 48 hours, resazurin (R&D Systems, Inc., Minneapolis, MN) was added and incubation resumed for four hours, then resorufin (the fluorescent reduction product of resazurin) fluorescence at 590 nm was measured as previously described. A four-parameter dose-response curve of resorufin fluorescence versus log(treatment) was generated to determine the ECso value for the IL-15-mediated metabolic activity.

A549 in vivo assay

The in vivo study was conducted in accordance with the Institutional Animal Care and Use Committee at the University of Minnesota. 3E6 A549/GFP/Luc in MATRIGEL (Corning, Inc., Coming, NY) diluted 3:2 with ice cold PBS were injected subcutaneously over the left ribcage of 8-12-week-old male or female NSG mice (^OD ^^-l ^} rkdc" ^cld II2r^ ^{( inlil ll} I z])' (day - 14). The tumors were allowed to engraft for 13 days, and initial tumor burden was assessed by bioluminescence imaging (BLI) on Day -1, with an imaging system (IVIS SPECTRUM, Xenogen, PerkinElmer, Waltham, MA) following intraperitoneal luciferin injection, to assign mice to treatment groups with equal tumor burden.

On Day 0, Day 7, and Day 14, 10 ⁶ ex vivo expanded NK cells were injected intravenously via the tail vein. 50 pg of either caml615TEM8 or functionally equivalent IL- 15 was injected intraperitoneally 5X per week for four weeks, then 3X per week thereafter. No treatment, equimolar IL-15, and caml615TEM8 in the absence of NK cells controls were also tested. Tumor length and width measurements were determined using a digital caliper (VWR International, LLC, Radnor, PA). Tumor volume was calculated as length *width ² /2.

On Day 28, mice were bled and RBCs from 100 pl of blood were lysed using ACK Lysing Buffer (Invitrogen, Waltham, MA; cat. no. A10492-01). Samples were stained with anti- CD3, anti-human CD45, anti-mouse CD45, and anti-CD56, and evaluated by flow cytometry for human NK cells. Tumors for histology were excised, placed in 10% formalin for 48 hours, then placed in 70% ethanol. Tumor samples were embedded into paraffin and stained with hematoxylin and eosin (H&E), anti-CD31. Whole slide digital scans were created with Zeiss Axio Scan.Zl and analyzed using QuPath vO.2.3 (Bankhead et al., Scientific Reports 7(1): 16878, 2017). Day 23 stained samples were inspected for the seven areas of the highest Granzyme B ⁺ cell density at 240X magnification. Day 42 stained samples were inspected for the seven areas of the highest CD31 ⁺ cell density at 120X magnification. A Granzyme B ⁺ cell detection mask was generated with the QuPath “Positive cell detection” analysis tool. The % CD31 ⁺ area was determined using image analysis with the QuPath software.

Data Analysis, Representation, and Statistical Analysis

PRISM (GraphPad Software, Inc., La lolla, CA) was used to generate graphs with error bars showing mean +/- SD (technical replicates) or SEM (biological replicates) and to calculate statistical significance, indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. PRISM software was used to calculate one-way ANOVA with Tukey’s multiple comparison tests when comparing three or more means and t tests when comparing two means. Logrank test for survival and four parameter logistic regression to calculate the half maximal effective concentration (EC50) was also performed using PRISM.

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Sequence Listing Free Text

SEQ ID NO: 1 - cam!6-IL15-anti-TEM8 sdAb (clone 9)

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL QESGGGLVQP GGSLRLSCAA SEYTFSSASM GWVRQAPGKG RREGVASIGF GGGSATYADS VKGRFTISRD NAKNTLYLQM NSLKPEDTAV YYCTSRFTYW GQGTQVTVSS cam 16: amino acids 1-122 flanking sequence: amino acids 123-144

IL-15: amino acids 145-258 flanking sequence: amino acids 259-276 anti-TEM8 sb Ab clone 9: amino acids 277-390

SEQ ID NO:2 - caml6-IL15-anti-TEM8 sdAb (clone 53)

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL QESGGGLVQP GGSLRLSCAA SGSTFEIASM GWVRQAPGKG REGVATIGSG GGYATYAESV KGRFTISRDN AKNTLYLQMN SLKPEDTAVY YCTSRYEYWG QGTQVTVSS cam 16: amino acids 1-122 flanking sequence: amino acids 123-144

IL-15: amino acids 145-258 flanking sequence: amino acids 259-276 anti-TEM8 sb Ab clone 53: amino acids 277-389

SEQ ID NO: 3 - caml6-IL15-anti-TEM8 sdAb (clone 89)

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL QESGGGLVQP GGSLRLSCAA SGRTFSSASM GWVRQAPGKG REGVAAIGFG GGSATYADSV KGRFTISRDN AKNTLYLQMN SLKPEDTAVY YCTSSYTSWG QGTQVTVSS cam 16: amino acids 1-122 flanking sequence: amino acids 123-144

IL-15: amino acids 145-258 flanking sequence: amino acids 259-276 anti-TEM8 sb Ab clone 89: amino acids 277-389

SEQ ID NO:4 - caml6-IL15-anti-TEM8 sdAb (clone 120)

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL QESGGGLVQP GGSLRLSCAA SGYTFSSAAM GWVRQAPGKG REGVAAIGSG EESATYAESV KGRFTISRDN AKNTLYLQMN SLKPEDTAVY YYTSDYEYWG QGTQVTVSS cam 16: amino acids 1-122 flanking sequence: amino acids 123-144

IL-15: amino acids 145-258 flanking sequence: amino acids 259-276 anti-TEM8 sbAb clone 120: amino acids 277-389

SEQ ID NO: 5 - cam!6-IL15-anti-TEM8 sdAb (clone 121)

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL QESGGGLVQP GGSLRLSCAA SGYTFSTAYM GWVRQAPGKG REGVASIGTG GEYATYADSV KGRFTISRDN AKNTLYLQMN SLKPEDTAVY YCTSTYEYWG QGTQVTVSS cam 16: amino acids 1-122 flanking sequence: amino acids 123-144

IL-15: amino acids 145-258 flanking sequence: amino acids 259-276 anti-TEM8 sbAb clone 121 : amino acids 277-389

SEQ ID NO: 6 - upstream crRNA spacer sequence

AATCTGGGAC AAAGAACCGT

SEQ ID NO: 7 - downstream crRNA spacer sequence

GCCTTTCCCA CCAACACGGG

SEQ ID NO:8 - (SGGG)4SG flanking sequence

SGGGGSGGGG SGGGGSGGGG SG SEQ ID NO: 9 - flanking sequence (Whitlow linker)

GSTSGSGKPG SGEGSTKG

SEQ ID NO: 10 - flanking sequence

EPKSSDKTHT SPPSPEL

SEQ ID NO: 11- flanking sequence

GGGGSGGGGS GGGGS

SEQ ID NO: 12- flanking sequence

PSGQAGAAAS ESLFVSNHAY

SEQ ID NO: 13- flanking sequence

EASGGPE

SEQ ID NO: 14 - flanking sequence

AEAAKEAAKE AAKEAAKALE AEAAKEAAKE AAKEAAKA

SEQ ID NO: 15- flanking sequence

AE AAKEAAKA

SEQ ID NO: 16- flanking sequence

SGGGGSGGGGS GGGGSGGGGSG

SEQ ID NO: 17- flanking sequence

GGGGSGGGGS

SEQ ID NO: 18 - flanking sequence

EPKSSDKTHT SPPSP

SEQ ID NO: 19- flanking sequence

RATPSHNSHQ VPSAGGPTAN SGTSG SEQ ID NO:20 - flanking sequence

SSGGGGSGGG GGGSSRSSL

SEQ ID NO:21 - camelid anti-CD16

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SS

SEQ ID NO:22 - IL-15

NWVNVISDLK KIEDLIQSMH IDATLYTESD VHPSCKVTAM KCFLLELQVI SLESGDASIH

DTVENLIILA NNSLSSNGNV TESGCKECEE LEEKNIKEFL QSFVHIVQMF INTS

SEQ ID NO:23- IL-12A

RNLPVATPDP GMFPCLHHSQ NLLRAVSNML QKARQTLEFY PCTSEEIDHE DITKDKTSTV EACLPLELTK NESCLNSRET SFITNGSCLA SRKTSFMMAL CLSSIYEDLK MYQVEFKTMN AKLLMDPKRQ I FLDQNMLAV IDELMQALNF NSETVPQKSS LEEPDFYKTK IKLCILLHAF RIRAVTIDRV MSYLNAS

SEQ ID NO:24 - IL-12B

IWELKKDVYV VELDWYPDAP GEMVVLTCDT PEEDGITWTL DQSSEVLGSG KTLTIQVKEF

GDAGQYTCHK GGEVLSHSLL LLHKKEDGIW STDILKDQKE PKNKTFLRCE AKNYSGRFTC

WWLTTISTDL TFSVKSSRGS SDPQGVTCGA ATLSAERVRG DNKEYEYSVE CQEDSACPAA EESLPIEVMV DAVHKLKYEN YTSSFFIRDI IKPDPPKNLQ LKPLKNSRQV EVSWEYPDTW STPHSYFSLT FCVQVQGKSK REKKDRVFTD KTSATVICRK NASISVRAQD RYYSSSWSEW ASVPCS

SEQ ID NO:25 - cam!615TEM8

QVQLVESGGG LVQPGGSLRL SCAASGLTFS SYNMGWFRQA PGQGLEAVAS ITWSGRDTFY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAANP WPVAAPRSGT YWGQGTLVTV SSSGGGGSGG GGSGGGGSGG GGSGNWVNVI SDLKKIEDLI QSMHIDATLY TESDVHPSCK VTAMKCFLLE LQVISLESGD ASIHDTVENL I ILANNSLSS NGNVTESGCK ECEELEEKNI KEFLQSFVHI VQMFINTSGS TSGSGKPGSG EGSTKGQVQL VQSGAEVKKP GTSVKVSCKV PGYTFSSYAI SWVRQAPGQG LEWMGGIIPI FGTTNYAQKF QGRVTITGEE STSTVYMELS SLRSEDTAVY YCARDTDYMF DYWGQGTLVT VSSGGGGSGG GGSGGGGSSS ELTQDPVVSV ALGETVSITC QGDNLRDFYA SWYQQKPGQA PLLVMYGKNR RPSGIPDRFS GSTSGNTLSL TITGAQAEDE ADYYCSSRDN SKHVVFGGGT KVTVL cam 16: amino acids 1-122 flanking sequence: amino acids 123-144 IL-15: amino acids 145-258 flanking sequence: amino acids 259-276 anti-TEM8 scFv: amino acids 277-515

VL domain: amino acids 277-393 linker: amino acids 394-408

VH domain: amino acids 409-515

SEQ ID NO:26 - TEM8 sdAb CDR1 (clone 9)

EYTFSSA

SEQ ID NO:27 - TEM8 sdAb CDR2 (clone 9)

GFGGGS

SEQ ID NO:28 - TEM8 sdAb CDR3 (clone 9)

RFTY

SEQ ID NO:29 - TEM8 sdAb CDR1 (clone 53)

GSTFEIA

SEQ ID NO:30 - TEM8 sdAb CDR2 (clone 53)

GSGGGY

SEQ ID NO:31 - TEM8 sdAb CDR3 (clone 53)

RYEY

SEQ ID NO:32 - TEM8 sdAb CDR1 (clone 89)

GRTFSSA

SEQ ID NO:27 - TEM8 sdAb CDR2 (clone 89); duplicate of clone 9 CDR2

GFGGGS

SEQ ID NO:33 - TEM8 sdAb CDR3 (clone 89)

SYTS SEQ ID NO:34 - TEM8 sdAb CDR1 (clone 120)

GYTFSSA

SEQ ID NO:35 - TEM8 sdAb CDR2 (clone 120)

GSGEES

SEQ ID NO:36 - TEM8 sdAb CDR3 (clone 120)

DYEY

SEQ ID NO:37 - TEM8 sdAb CDR1 (clone 121)

GYTFSTA

SEQ ID NO:38 - TEM8 sdAb CDR2 (clone 121)

GTGGEY

SEQ ID NO:39 - TEM8 sdAb CDR3 (clone 121)

TYEY

SEQ ID NO:40 - camelid anti-CD16 CDR1

GLTFSSY

SEQ ID NO:41 - camelid anti-CD16 CDR2

TWSGRDT

SEQ ID NO:42 - camelid anti-CD16 CDR3

NPWPVAAPRS GTY